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The aim of this study was to characterize the mutational landscape of patients with FLT3-mutated acute myeloid leukemia (AML) treated within the randomized CALGB 10603/RATIFY trial evaluating intensive chemotherapy plus the multi-kinase inhibitor midostaurin versus placebo. We performed sequencing of 262 genes in 475 patients: mutations occurring concurrently with the FLT3-mutation were most frequent in NPM1 (61%), DNMT3A (39%), WT1 (21%), TET2 (12%), NRAS (11%), RUNX1 (11%), PTPN11 (10%), and ASXL1 (8%) genes. To assess effects of clinical and genetic features and their possible interactions, we fitted random survival forests and interpreted the resulting variable importance. Highest prognostic impact was found for WT1 and NPM1 mutations, followed by white blood cell count, FLT3 mutation type (internal tandem duplications vs. tyrosine kinase domain mutations), treatment (midostaurin vs. placebo), ASXL1 mutation, and ECOG performance status. When evaluating two-fold variable combinations the most striking effects were found for WT1:NPM1 (with NPM1 mutation abrogating the negative effect of WT1 mutation), and for WT1:treatment (with midostaurin exerting a beneficial effect in WT1-mutated AML). This targeted gene sequencing study provides important, novel insights into the genomic background of FLT3-mutated AML including the prognostic impact of co-mutations, specific gene–gene interactions, and possible treatment effects of midostaurin.

Publisher Copyright: © 2022 Okoye-Okafor et al. Thrombocytopenia, prevalent in the majority of patients with myeloid malignancies, such as myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML), is an independent adverse prognostic factor. Azacitidine (AZA), a mainstay therapeutic agent for stem cell transplant–ineligible patients with MDS/AML, often transiently induces or further aggravates disease-associated thrombocytopenia by an unknown mechanism. Here, we uncover the critical role of an acute type-I interferon (IFN-I) signaling activation in suppressing megakaryopoiesis in AZA-mediated thrombocytopenia. We demonstrate that megakaryocytic lineage-primed progenitors present IFN-I receptors and, upon AZA exposure, engage STAT1/SOCS1-dependent downstream signaling prematurely attenuating thrombopoietin receptor (TPO-R) signaling and constraining megakaryocytic progenitor cell growth and differentiation following TPO-R stimulation. Our findings directly implicate RNA demethylation and IFN-I signal activation as a root cause for AZA-mediated thrombocytopenia and suggest mitigation of TPO-R inhibitory innate immune signaling as a suitable therapeutic strategy to support platelet production, particularly during the early phases of AZA therapy.

Next generation sequencing studies in Chronic lymphocytic leukemia (CLL) have revealed novel genetic variants that have been associated with disease characteristics and outcome. The aim of this study was to evaluate the prognostic value of recurrent molecular abnormalities in patients with CLL. Therefore, we assessed their incidences and associations with other clinical and genetic markers in the prospective multicenter COMPLEMENT1 trial (treatment naive patients not eligible for intensive treatment randomized to chlorambucil (CHL) vs. ofatumumab-CHL (O-CHL)). Baseline samples were available from 383 patients (85.6%) representative of the total trial cohort. Mutations were analyzed by amplicon-based targeted next generation sequencing (tNGS). In 52.2% of patients we found at least one mutation and the incidence was highest in NOTCH1 (17.0%), followed by SF3B1 (14.1%), ATM (11.7%), TP53 (10.2%), POT1 (7.0%), RPS15 (4.4%), FBXW7 (3.4%), MYD88 (2.6%) and BIRC3 (2.3%). While most mutations lacked prognostic significance, TP53 (HR2.02,p

The immunologic microenvironment in various solid tumors is aberrant and correlates with clinical survival. Here, we present a comprehensive analysis of the immune environment of acute myeloid leukemia (AML) bone marrow (BM) at diagnosis. We compared the immunologic landscape of formalin-fixed paraffin-embedded BM trephine samples from AML (n = 69), chronic myeloid leukemia (CML; n = 56), and B-cell acute lymphoblastic leukemia (B-ALL) patients (n = 52) at diagnosis to controls (n = 12) with 30 immunophenotype markers using multiplex immunohistochemistry and computerized image analysis. We identified distinct immunologic profiles specific for leukemia subtypes and controls enabling accurate classification of AML (area under the curve [AUC] = 1.0), CML (AUC = 0.99), B-ALL (AUC = 0.96), and control subjects (AUC = 1.0). Interestingly, 2 major immunologic AML clusters differing in age, T-cell receptor clonality, and survival were discovered. A low proportion of regulatory T cells and pSTAT1+cMAF− monocytes were identified as novel biomarkers of superior event-free survival in intensively treated AML patients. Moreover, we demonstrated that AML BM and peripheral blood samples are dissimilar in terms of immune cell phenotypes. To conclude, our study shows that the immunologic landscape considerably varies by leukemia subtype suggesting disease-specific immunoregulation. Furthermore, the association of the AML immune microenvironment with clinical parameters suggests a rationale for including immunologic parameters to improve disease classification or even patient risk stratification.

Contains fulltext : 218279.pdf (Publisher’s version ) (Open Access) Patients with acute myeloid leukemia (AML) harboring FLT3 internal tandem duplications (ITDs) have poor outcomes, in particular AML with a high (>/=0.5) mutant/wild-type allelic ratio (AR). The 2017 European LeukemiaNet (ELN) recommendations defined 4 distinct FLT3-ITD genotypes based on the ITD AR and the NPM1 mutational status. In this retrospective exploratory study, we investigated the prognostic and predictive impact of the NPM1/FLT3-ITD genotypes categorized according to the 2017 ELN risk groups in patients randomized within the RATIFY trial, which evaluated the addition of midostaurin to standard chemotherapy. The 4 NPM1/FLT3-ITD genotypes differed significantly with regard to clinical and concurrent genetic features. Complete ELN risk categorization could be done in 318 of 549 trial patients with FLT3-ITD AML. Significant factors for response after 1 or 2 induction cycles were ELN risk group and white blood cell (WBC) counts; treatment with midostaurin had no influence. Overall survival (OS) differed significantly among ELN risk groups, with estimated 5-year OS probabilities of 0.63, 0.43, and 0.33 for favorable-, intermediate-, and adverse-risk groups, respectively (P < .001). A multivariate Cox model for OS using allogeneic hematopoietic cell transplantation (HCT) in first complete remission as a time-dependent variable revealed treatment with midostaurin, allogeneic HCT, ELN favorable-risk group, and lower WBC counts as significant favorable factors. In this model, there was a consistent beneficial effect of midostaurin across ELN risk groups.

The results from the RATIFY trial (ClinicalTrials.gov: NCT00651261; CALGB 10603) showed that midostaurin combined with standard chemotherapy significantly improved outcomes in patients with FMS-like tyrosine kinase 3 (FLT3)–mutated acute myeloid leukemia (AML), compared with placebo. In this post hoc subgroup analysis from the trial, we evaluated the impact of midostaurin in 163 patients with FLT3-tyrosine kinase domain (TKD) mutations. At a median follow-up of 60.7 months (95% CI, 55.0-70.8), the 5-year event-free survival (EFS) rate was significantly higher in patients treated with midostaurin than in those treated with placebo (45.2% vs 30.1%; P = .044). A trend toward improved disease-free survival was also observed with midostaurin (67.3% vs 53.4%; P = .089), whereas overall survival (OS) was similar in the 2 groups. Patients with AML and NPM1mut/FLT3-TKDmut or core binding factor (CBF)–rearranged/FLT3-TKDmut genotypes had significantly prolonged OS with or without censoring at hematopoietic cell transplantation (HCT), compared with NPM1WT/CBF-negative AMLs. The multivariable model for OS and EFS adjusted for allogeneic HCT in first complete remission as a time-dependent covariable, revealed NPM1 mutations and CBF rearrangements as significant favorable factors. These data show that NPM1 mutations or CBF rearrangements identify favorable prognostic groups in patients with FLT3-TKD AMLs, independent of other factors, also in the context of midostaurin treatment.

Thrombocytopenia defined as platelet counts of Several studies evaluating the use of thrombopoietin receptor agonists (TPO-RA) for the clinical management of thrombocytopenia have shown promising clinical results. TPO-RA eltrombopag (EP) which has been effective as a single agent to raise platelet counts in MDS, but failed to stimulate platelet production in a phase III placebo-controlled clinical study when used in combination with AZA (NCT02158936). Here, we assessed the molecular and cellular mechanisms of AZA contributing to thrombocytopenia and interfering with TPO-RA mediated rescue. Our results demonstrate that AZA mediates the rapid induction of dsRNAs and activation of interferon type I (IFN-I) signaling in various hematopoietic cells, including stem and progenitor cells of healthy donors and MDS/AML. This engagement of IFN-I/STAT1/SOCS1 signaling resulted in significant inhibition of megakaryocytic progenitor growth and differentiation, independently of DNA hypomethylation and endogenous retroviruses. We show that the inhibitory effects of AZA on megakaryopoeisis can be counteracted through inhibition of IFN-I signaling, SOCS1 activation or P38 MAPK activity. Our findings provide evidence intercepting inhibition of TPO-R signaling by AZA-induced innate immune IFN-I pathway and thrombocytopenia.

Eltrombopag (EP), a small molecule thrombopoietin receptor (TPO-R) agonist and potent intracellular iron chelator, has shown remarkable efficacy in stimulating sustained multilineage hematopoiesis in patients with bone marrow failure syndromes, suggesting an effect at the most immature hematopoietic stem and multipotent progenitor level. Although the functional and molecular effects of EP on megakaryopoiesis have been studied in the past, mechanistic insights into its effects on the earliest stages of hematopoiesis have been limited. Here, we investigated the effects of EP treatment on hematopoietic stem cell (HSC) function using purified primary HSCs in separation-of-function mouse models, including a TPO receptor-deficient strain, and stem cells isolated from patients undergoing TPO-R agonist treatment. Our mechanistic studies showed a stimulatory effect on stem cell self-renewal independently of TPO-R. Human and mouse HSCs responded to acute EP treatment with metabolic and gene expression alterations consistent with a reduction of intracellular labile iron pools that are essential for stem cell maintenance. Iron preloading prevented the stem cell-stimulatory effects of EP. Moreover, comparative analysis of stem cells in the bone marrow of patients receiving EP showed a marked increase in the number of functional stem cells compared to patients undergoing therapy with romiplostim, another TPO-R agonist lacking an iron-chelating ability. Taken altogether, our study demonstrates that EP stimulates hematopoiesis at the stem cell level through iron chelation-mediated molecular reprograming and indicates that labile iron pool-regulated pathways can modulate hematopoietic stem cell function.

To the editor: The anti-CD38 monoclonal antibody (mAb) daratumumab is effective in multiple myeloma (MM) and is increasingly being used at first relapse in combination with bortezomib/dexamethasone or lenalidomide/dexamethasone, which are capable of inducing complete responses in a notable

Sunitinib is a tyrosine kinase inhibitor approved for the treatment of multiple solid tumors. However, cardiotoxicity is of increasing concern, with a need to develop rational mechanism driven approaches for the early detection of cardiac dysfunction. We sought to interrogate changes in cardiac energy substrate usage during sunitinib treatment, hypothesising that these changes could represent a strategy for the early detection of cardiotoxicity. Balb/CJ mice or Sprague-Dawley rats were treated orally for 4 weeks with 40 or 20 mg/kg/day sunitinib. Cardiac positron emission tomography (PET) was implemented to investigate alterations in myocardial glucose and oxidative metabolism. Following treatment, blood pressure increased, and left ventricular ejection fraction decreased. Cardiac [18F]-fluorodeoxyglucose (FDG)-PET revealed increased glucose uptake after 48 hours. [11C]Acetate-PET showed decreased myocardial perfusion following treatment. Electron microscopy revealed significant lipid accumulation in the myocardium. Proteomic analyses indicated that oxidative metabolism, fatty acid β-oxidation and mitochondrial dysfunction were among the top myocardial signalling pathways perturbed. Sunitinib treatment results in an increased reliance on glycolysis, increased myocardial lipid deposition and perturbed mitochondrial function, indicative of a fundamental energy crisis resulting in compromised myocardial energy metabolism and function. Our findings suggest that a cardiac PET strategy may represent a rational approach to non-invasively monitor metabolic pathway remodeling following sunitinib treatment.

Objectives ABIGAIL, a phase II, randomized, open-label, multicenter study evaluated the correlation between biomarkers and best overall response (BOR) to bevacizumab with chemotherapy in patients with advanced or recurrent non-small-cell lung cancer (NSCLC). Exploratory analyses of vascular endothelial growth factor (VEGF) clinical genotyping data are presented. Materials and methods A total of 303 patients with NSCLC were randomized to receive bevacizumab 7.5 mg/kg or 15 mg/kg until progression or unacceptable toxicity (plus six cycles of chemotherapy). Patients provided blood samples for biomarker analysis. Exploratory analyses were conducted to assess whether genetic variants in VEGF-A or VEGFR-1/-2 act as efficacy or safety biomarkers. Single nucleotide polymorphisms (SNPs) were determined using individual genotyping assays. DNA analysis for 12 SNPs across three genes is reported: VEGF-A (five SNPs), VEGFR-1 (three SNPs), and VEGFR-2 (four SNPs). Results VEGF-A: c.+405/c.−634 (CG), VEGF-A: c.−460 >C; c−1498 >C (CT), and VEGF-A: c.−2578 C>A were associated with >50% higher odds of responding to treatment. VEGFR-1: rs9554316 (GT) was associated with >30% higher risk of progression and >40% higher risk of death. VEGF-A: c.+936 C>T was associated with higher incidence of hypertension. Conclusions Four genetic variants of VEGF-A and VEGFR-1 were associated with bevacizumab treatment outcome. Three variants in VEGF-A were associated with increased BOR, one variant in VEGFR-1 was associated with worse progression-free survival/overall survival. These associations were not statistically significant after correction for multiple testing. No genetic variant was associated with significantly higher risk of hypertension. Replication in additional studies may provide insight into the use of these variants to predict response to bevacizumab.

Despite extensive translational research, no validated biomarkers predictive of bevacizumab treatment outcome have been identified.We performed a meta-analysis of individual patient data from six randomized phase III trials in colorectal, pancreatic, lung, renal, breast, and gastric cancer to explore the potential relationships between 195 common genetic variants in the vascular endothelial growth factor (VEGF) pathway and bevacizumab treatment outcome.The analysis included 1,402 patients (716 bevacizumab-treated and 686 placebo-treated). Twenty variants were associated (P0.05) with progression-free survival (PFS) in bevacizumab-treated patients. Of these, 4 variants in EPAS1 survived correction for multiple testing (q0.05). Genotype-by-treatment interaction tests revealed that, across these 20 variants, 3 variants in VEGF-C (rs12510099), EPAS1 (rs4953344), and IL8RA (rs2234671) were potentially predictive (P0.05), but not resistant to multiple testing (q0.05). A weak genotype-by-treatment interaction effect was also observed for rs699946 in VEGF-A, whereas Bayesian genewise analysis revealed that genetic variability in VHL was associated with PFS in the bevacizumab arm (q0.05). Variants in VEGF-A, EPAS1, and VHL were located in expression quantitative loci derived from lymphoblastoid cell lines, indicating that they affect the expression levels of their respective gene.This large genetic analysis suggests that variants in VEGF-A, EPAS1, IL8RA, VHL, and VEGF-C have potential value in predicting bevacizumab treatment outcome across tumor types. Although these associations did not survive correction for multiple testing in a genotype-by-interaction analysis, they are among the strongest predictive effects reported to date for genetic variants and bevacizumab efficacy.

Management of hematologic disease- and therapy-related thrombocytopenia remains a serious clinical issue, especially in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The ribonucleoside and DNA-demethylating agent azacytidine (AZA), has proven useful for the treatment of patients with MDS and AML not eligible for stem cell transplantation. While low-dose AZA therapy induces clinical remissions in up to 50% of treated patients, it comes at the cost of aggravating pre-existing thrombocytopenia which is observed in a subset of patients; this can lead to increased bleeding and bleeding-associated mortality, and importantly, often requires dose modifications and delays of therapy. Thus, identification of strategies alleviating ineffective megakaryopoiesis will likely lead to increased therapeutic efficacy for patients with MDS/AML. Eltrombopag (EP), a second-generation small molecule thrombopoietin receptor (TPO-R) agonist was effective in raising platelet counts in patients with MDS as a single agent, as well as in combination with certain standard of care therapies. However, it failed to stimulate platelet production during the first four cycles of AZA treatment as uncovered by a recent phase III placebo-controlled clinical study (SUPPORT; NCT02158936). The goals of this study were to identify the cellular and molecular underpinnings of AZA-associated inhibition of megakaryopoiesis and to assess the ineffectiveness of EP in mitigating AZA treatment-associated thrombocytopenia. Our results demonstrate that at a clinically-equivalent and non-cytotoxic dose, AZA rapidly induces transient activation of interferon type I (IFN-I) signaling in various hematopoietic cell types, including stem and lineage-committed progenitor cells (HSPCs). We detected IFNα and IFNβ production and release using ELISA and intracellular flow cytometry on primary total mononuclear cell- and purified CD34-positive HSPC populations derived from cord blood, bone marrow from healthy volunteers or patients with MDS/AML. AZA-mediated activation of Type I IFNs in healthy control- and MDS/AML cells was preceded by an accumulation of double-stranded RNA (dsRNA) species and decreased total RNA cytosine methylation measured by immunocytochemistry and intracellular FACS analysis; this suggested that AZA triggered the accumulation of immunogenic RNA species which elicit an IFN-I response. In support, we found Toll like receptor 3 (TLR3) activation and phosphorylation of STAT1 in CD34+ HPSC, along with premature activation of Suppressor of Cytokine Signaling 1 (SOCS1), a well-known JAK/STAT-dependent signaling attenuator. This rapid AZA-induced viral mimicry response led to abrogation of thrombopoietin (TPO) or EP-stimulated TPO-R signaling and inhibition of ex vivo megakaryocyte progenitor proliferation quantified by colony formation in semi-solid medium. Importantly, inhibition of IFN-I signal activation using the JAK3 inhibitor decernotinib, the IFNα/β-blocking peptide, B18R, or RNA interference-mediated knock-down of SOCS1 counteracted the inhibitory effects of AZA on TPO-R stimulation and restored megakaryopoiesis. Given these observations, we pre-clinically tested a revised treatment protocol, in which primary cells were first exposed to AZA for four days followed by TPO-R stimulation using TPO or EP. This new treatment strategy alleviated AZA's inhibitory effects at the molecular and cellular levels, demonstrating that upon resolution of the AZA-mediated vial mimicry response, EP and TPO can effectively stimulate TPO-R signaling and megakaryopoiesis. Together, our data reveal a mechanistic basis of AZA-mediated inhibition of megakaryopoiesis in patients with MDS/AML. Additionally, we show that EP cannot overcome the megakaryopoiesis-inhibitory effects of acute IFN-I signaling activation upon AZA exposure. Findings of our study are consistent with and provide a molecular explanation for the observations made in the context of the SUPPORT study. In the future, it will be critical to better understand and potentially counteract the megakaryopoiesis-inhibitory effects by IFN-I pathway activation upon AZA therapy in patients with MDS/AML. Disclosures Okoye-Okafor: Novartis Pharmaceuticals: Research Funding. Pallaud:Novartis Pharmaceuticals: Employment. Marques Ramos:Novartis Pharmaceuticals: Employment. Verma:Janssen: Research Funding; BMS: Research Funding; Celgene: Honoraria; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding. Will:Novartis Pharmaceuticals: Research Funding.

Background: Several candidate BMs have been explored in randomized trials of BEV across tumor types with the aim of identifying patients (pts) deriving the most substantial benefit from BEV therapy. In phase III trials, baseline pVEGF-A and pVEGFR-2 showed potential predictive value in metastatic BC (AVADO, AVEREL), pancreatic cancer (AViTA), and gastric cancer (AVAGAST; VEGF-A only). The randomized phase III BEATRICE trial, evaluating the addition of BEV to adjuvant chemotherapy in pts with triple-negative early BC, includes a comprehensive program to identify potential BMs predicting efficacy and toxicity of BEV therapy. We report results for baseline pVEGF-A and pVEGFR-2. Methods: After selection of chemotherapy (anthracycline and/or taxane), pts with T1a-T3 operable BC were randomized 1:1 to receive ≥4 cycles of chemotherapy either alone or with 1 year of BEV 5 mg/kg/wk equivalent. The primary endpoint is invasive disease-free survival (IDFS). pEDTA samples were collected from consenting pts at baseline (before treatment, after surgery), during study treatment, and at relapse. Pts were dichotomized using the median baseline concentration of each marker as the cut-off between high and low cohorts; further exploratory analyses were also performed by quartile. Results: Between Dec 2007 and Mar 2010, 2591 pts were enrolled. Of these, 1273 (49%) consented to the BM study and 1178 (45%) were included in the BM-evaluable population (BEP). Overall, the BEP was representative of the ITT population except for lower proportions of Asian pts (12% vs 24%). IDFS was similar in the BEP and ITT populations. Baseline characteristics were balanced between arms in the BEP. Baseline pVEGF-A showed neither prognostic nor predictive value using the median as the cut-off, although with a third quartile (Q3) cut-off there was a more pronounced but non-significant differentiation between treatments (HR 0.92 [low] vs 0.64 [high]). High baseline pVEGFR-2 showed potential predictive value for BEV efficacy (HR 1.24 [low] vs 0.61 [high]; p=0.029). Conclusions: Unlike trials in metastatic BC (AVADO, AVEREL), in the adjuvant setting, baseline pVEGF-A concentration did not show predictive value for BEV efficacy with a median cut-off. However, analyses using the Q3 cut-off suggest a trend toward predictive value. High baseline pVEGFR-2 was associated with greater BEV treatment effect, consistent with previous results in AVADO and AVEREL. The impact of differing biology in the adjuvant setting, lower median VEGF-A concentration than in the metastatic setting (77.0 vs 125.0–129.1 pg/mL), and the possible influence of surgery immediately before treatment require further investigation. Additional exploratory analyses are ongoing to provide better understanding of the BEATRICE dataset and the complex biology of angiogenesis, including additional markers, changes over time, and combination signatures. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-06-34.

Background Following the positive outcome of the RATIFY phase 3 clinical trial, the multi-kinase inhibitor midostaurin was approved for the treatment of adult patients with newly diagnosed FLT3-mutated acute myeloid leukemia (AML). However, we and others have observed that single agent midostaurin yields responses also in a substantial portion of patients not carrying FLT3 mutations. The molecular basis and the kinase targets mediating these responses are poorly understood and no biomarkers predictive of response in FLT3 wildtype (wt) AML patients exist. To identify markers distinguishing the FLT3 wt responding subset of patients, we trained machine learning multi-marker models using AML patient baseline transcriptomic and mutational data to predict ex vivo responders vs. non-responders. Further, to better understand the molecular basis of midostaurin responses and to explore the unique signaling networks modulated by midostaurin, we profiled the sensitivities of AML patient samples to midostaurin in comparison to, and in combination with, several clinically relevant oncological targeted agents of diverse mechanistic classes. Results Midostaurin target space is unique and it retains anti-leukemic potency under cytoprotective conditions. We have previously established that single agent midostaurin is effective ex vivo in about 25% of FLT3 wt AML patient samples and retains potency in a cytoprotective medium that masks the effects of more selective FLT3 inhibitors such as quizartinib, crenolanib and sorafenib (Karjalainen et al, Blood 2017). To further investigate the unique pathways that midostaurin, but not other FLT3 inhibitors targets, we correlated the response patterns of 87 AML patient samples in cytoprotective medium to midostaurin and 261 other kinase inhibitors in our oncology compound collection. In unsupervised cluster analysis, midostaurin showed highly similar response patterns to AZD7762, OTS167, milciclib, pacritinib, ENMD-2076 and fostamatinib. Publicly available in vitro kinase profiling (Tang et al, Cell Chem. Biol. 2018) suggested that midostaurin does not inhibit most of the primary targets of these other inhibitors, with only aurora kinases, JAK kinases and SYK appearing to be shared potent targets. Midostaurin anti-leukemic potency is determined by the mutational background. Several multi-marker, supervised machine learning models were compared to extract biomarker signatures from either baseline transcriptomic or mutational data, in the task of predicting ex vivo midostaurin response in samples cultured in cytoprotective medium. In the full cohort (N=81), the presence of FLT3 mutations (both internal tandem repeat and tyrosine kinase domain mutations) was the strongest predictor of response. In the FLT3 wt cases (N=49), our results revealed that other select mutations correlated well with either response or non-response upon Bayesian Linear Regression analysis with cross-validation (Ammad-Ud-Din et al, Bioinformatics, 2017). Mutations in PTPN11, U2AF1, SRSF2, RUNX1, JAK2 and BCOR predicted midostaurin responders, while mutations in GATA2, WT1, NPM1 and IDH2 were enriched in non-responders (Figure 1). Baseline transcriptomic profiles, however, did not provide added value for the predictive power. Midostaurin efficacy can be enhanced by combination with other targeted agents. Combinatorial drug screening of midostaurin in cytoprotective medium revealed several synergizing drug classes, including BCL-2 and MDM2-p53 inhibitors. Further analysis of synergizing agents in broader AML patient sample cohorts is ongoing. Conclusions Our results show that midostaurin may reach its biological effects through inhibition of additional kinases than just FLT3. In both FLT3 mutant and wt cases, midostaurin responses are influenced by the overall mutational background. Furthermore, our data indicates that midostaurin efficacy can be enhanced through combination with other agents. Together, we have significantly expanded the understanding of molecular determinants of midostaurin response in primary AML cells, supporting predictive biomarker discovery efforts and development of synergistic drug combinations. The emerging hypotheses from this work will have to be tested in clinical studies. Disclosures Porkka: Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Marques Ramos:Novartis: Employment. Pallaud:Novartis: Employment. Aittokallio:Novartis: Research Funding. Wennerberg:Novartis: Research Funding.

Background: Recently, the oral multitargeted small molecule FLT3 inhibitor midostaurin (M) was approved for treatment of FLT3-mutated AML in combination with standard chemotherapy. In the international RATIFY (NCT00651261) trial, addition of M led to superior overall and event-free survival compared to placebo, thus defining a new standard of care in this AML subset (Stone RM et al. NEJM 2017). Although not powered for subgroup analyses, M showed consistent effects across all FLT3 mutation strata [tyrosine kinase domain (TKD); internal tandem duplication (ITD) with low (0.05-0.7; ITDlow) or high (>0.7; ITDhigh) allelic ratio] suggesting significant off-target activity beyond FLT3 inhibition. Aim: We aimed to comprehensively profile the mutational landscape of FLT3 mutated (FLT3mut) AML in a large, well characterized cohort of patients (pts) treated within the RATIFY trial using a high-throughput targeted sequencing (HTS) approach. Methods: HTS was performed on the entire coding region of 262 genes involved in hematologic malignancies including 20 genes that encode kinases targeted by M (M kinome, MK). Pretreatment peripheral blood (PB; 14%) or bone marrow (BM; 86%) specimens were available from 475 (66%) of 717 FLT3mut AML RATIFY pts. Libraries were prepared using SureSelectXT custom solutions (Agilent). Paired-end sequencing was carried out on a HiSeq platform (Illumina). FLT3 mutation (mut) status was available for all pts [TKD: 24%; ITD: 76% (ITDlow: 45%; ITDhigh:31%)], and cytogenetic data for 454 pts (96%). Results: An average sequencing depth of 978x was obtained for all pts. In sum, 1815 mut (missense: 49%; indels: 40%; nonsense: 7%; other: 3%) were identified with a mean of 3.8 mut per pt (FLT3 strata; TKD: 4; ITDlow: 4; ITDhigh: 3.6).Overall, recurrent mut (>5% of all pts) were found in NPM1 (61%), DNMT3A (39%), WT1 (21%), TET2 (12%), RUNX1 (11%), NRAS (11%), PTPN11 (9%), ASXL1 (8%), IDH1 (8%), IDH2 (7%; R140 only), and SMC1A (6%). In contrast, TP53 (1%) and biallelic CEPBA (1%) mut were rare events. This was also true for aberrations of the MK (7% in total) with KIT (2%), MAP3K11 (1%), and NTRK3 (1%) being most frequently mutated. When stratified according to FLT3mut type, mut in NRAS (24% vs 7%, p Based on the mut and cytogenetic data, we next sought to assign all FLT3mut pts to the 11 recently defined molecular AML classes (Papaemmanuil E et al. NEJM 2016). The majority fell into two classes, namely the NPM1 (N; 62%) and the chromatin-spliceosome (CS; 15%) classes, underscoring the significance of FLT3mut as the driver in these particular genomic classes. Other class-defining lesions were rare or absent in this cohort [inv(16): 2%; t(8;21): 2%; t(11q23;x): 2%; t(6;9): 1%, TP53-aneuploidy: 1%; CEBPAbiallelic: 1%; IDH2R172: 0%]. In 14% of all pts categorization was not possible (no or >1 class-defining lesion), emphasizing the need for further refinement of this classification. When focusing on these two groups, N and CS had comparable FLT3mut patterns (TKD: 24% vs 21%; ITDlow: 44% vs 45%; ITDhigh: 32% vs 33%), whereas N more frequently correlated with a normal karyotype (N: 91% vs CS: 63%). With respect to clinical characteristics, no differences between N and CS in terms of age, white blood cells, platelets, PB and BM blasts, as well as history of MDS were observed. Conclusion: In this comprehensive sequencing approach, we could further delineate the molecular pattern of FLT3mut AML. Here, FLT3-ITD and -TKD groups exhibited remarkable differences in cooperating pathways, highlighting distinct biologic features in the leukemogenesis of FLT3mut AML. Overall, mut of MK genes were rare events, not fully explaining the complexity of M off-target effects. Understanding the underlying disease mechanism will potentially provide useful information on prognosis and prediction of response to M. Further analyses including correlation with clinical outcome are ongoing. Support: U10CA180821, U10CA180861, U10CA180882, U24CA196171 Disclosures Bullinger: Janssen: Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Pfizer: Speakers Bureau; Sanofi: Research Funding, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Bayer Oncology: Research Funding. Gathmann:Novartis: Employment. Larson:Ariad/Takeda: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BristolMyers Squibb: Consultancy, Research Funding. Medeiros:Genentech: Employment; Celgene: Consultancy, Research Funding. Tallman:ADC Therapeutics: Research Funding; AROG: Research Funding; BioSight: Other: Advisory board; Orsenix: Other: Advisory board; AbbVie: Research Funding; Daiichi-Sankyo: Other: Advisory board; Cellerant: Research Funding. Tiecke:Novartis: Employment. Pallaud:Novartis: Employment. Ehninger:Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Bayer: Research Funding. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees. Stone:Otsuka: Consultancy; Jazz: Consultancy; Cornerstone: Consultancy; Fujifilm: Consultancy; Arog: Consultancy, Research Funding; Pfizer: Consultancy; Sumitomo: Consultancy; Novartis: Consultancy, Research Funding; Ono: Consultancy; Orsenix: Consultancy; Merck: Consultancy; Argenx: Other: Data and Safety Monitoring Board; AbbVie: Consultancy; Agios: Consultancy, Research Funding; Amgen: Consultancy; Astellas: Consultancy; Celgene: Consultancy, Other: Data and Safety Monitoring Board, Steering Committee. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding. Döhner:AROG Pharmaceuticals: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AROG Pharmaceuticals: Research Funding; Pfizer: Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celator: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Jazz: Consultancy, Honoraria.

Introduction: Internal tandem duplication of the FLT3 gene (FLT3-ITD), resulting in duplication of 3 to more than hundreds of nucleotides, are present in approximately 25% of adults with newly diagnosed AML. Several studies have shown that ITD mutations are associated with poor prognosis due to a high relapse rate, in particular in case of a high mutant to wild-type allele ratio and/or insertion site in the beta1-sheet of the tyrosine kinase domain-1 (beta1-sheet). Aims: To investigate the relationship between ITD insertion site and patient outcome, Roche 454 next generation sequencing (NGS) was performed in 452/555 (81.4%) FLT3-ITD positive patients (pts) enrolled into the RATIFY trial (NCT00651261). Results: NGS identified 908 ITDs with up to 9 ITDs per case (1 ITD: n=210, 46.5%; 2 ITDs: n=131, 29.0%; 3 ITDs: n=58, 12.8%; 4 ITDs: n=24, 5.3%; 5 ITDs: n=18, 4.0%; 6 ITDs: n=3, 0.7%; 7 ITDs: n=7, 1.5%; 9 ITDs: n=1, 0.2%). Median ITD-size was 45 nucleotides (range, 6-246); all ITDs were in-frame with direct head-to-tail orientation. According to the 4 functional groups, 488 ITDs (53.7%) were located within the juxtamembrane domain (JMD), 155 ITDs (17.1%) within the hinge region, 211 ITDs (23.2%) within the beta1-sheet, and 54 ITDs (5.9%) 3´of beta1-sheet. ITD size strongly correlated with insertion site, in that the more C-terminal the insertion site, the longer the size of the inserted fragment (P NPM1 mutations (NPM1mut) were present in 203/358 pts (56.7%). Correlation of ITD insertion site with NPM1mut revealed a significantly lower incidence of NPM1mut in pts with insertion located within the hinge region (50/106, 47.2% vs 153/252, 60.7%; P=.02) and 3´of beta1-sheet (14/41, 34.1% vs 189/317, 59.6%; P=.002), whereas NPM1mut were significantly more frequent in pts with insertions affecting JMD (143/235, 60.9% vs 60/123, 48.8%; P=.03). Clinical characteristics differing among the 4 functional ITD groups were gender and WBC. Pts with insertions 3´of beta1-sheet were predominantly male (28/46, 60.9% vs 178/406, 43.8%; P=.03); pts with JMD insertions exhibited lower WBC (median 36.5 vs 52.7 x109/L; P=.03). Complete remission (CR) was achieved within 60 days in 248/452 pts (54.9%). To evaluate the impact of ITD insertion site on response to induction, a logistic regression model was used. ITD insertion sites were categorized in (i) only in beta1-sheet, (ii) in beta1-sheet and other sites, and (iii) outside the beta1-sheet. Other variables were ITD mutant to wild-type allelic ratio (fragment analysis, cutoff at 0.5), number of ITDs per patient, log2 of WBC counts, age, NPM1mut, and midostaurin treatment. In this model, only number of ITDs predicted lower CR rate (OR, 0.72; 95% CI, 0.57-0.90), while NPM1mut was a favorable marker for CR (OR, 2.69; 95% CI, 1.70-4.28). Median follow-up for survival was 60.6 months (mo); median event free survival (EFS) and overall survival (OS) were 3.9 mo and 24.4 mo, respectively. The 4-year EFS and OS rates were 21.0% (95% CI, 17.2%-24.8%) and 42.6% (95% CI, 37.9%-47.4%), respectively. Survival analysis according to categorized insertion site groups showed that pts exhibiting insertion exclusively in the beta1-sheet had significantly inferior OS (P=.014) compared to the other two groups. Multivariate models for OS and EFS including hematopoietic stem-cell transplantation (HSCT) as a time-dependent covariate revealed WBC counts as unfavorable and NPM1mut as favorable for both endpoints; further unfavorable factors were older age and exclusive insertion in the beta1-sheet (HR, 1.49; 95% CI, 1.01-2.20) for OS and number of ITDs (HR, 1.15; 95% CI, 1.04-1.28) for EFS; HSCT was a favorable factor only for EFS (HR, 0.66; 95% CI, 0.44-0.99). Midostaurin treatment was associated with in trend improved EFS (HR, 0.81; 95% CI, 0.63-1.03) and OS (HR, 0.77; 95% CI, 0.57-1.02). Conclusions: In this large cohort of 452 FLT3-ITD mutated AML treated within the RATIFY trial the negative prognostic impact of beta1-sheet insertion site of FLT3-ITD could be confirmed. Further analyses to investigate potential predictive effects of midostaurin treatment are ongoing. Support: U10CA180821, U10CA180882, U24CA196171, (CCSRI) #704970. Disclosures Du: Novartis: Employment. Gathmann:Novartis: Employment. Larson:Ariad/Takeda: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; BristolMyers Squibb: Consultancy, Research Funding. Medeiros:Celgene: Consultancy, Research Funding; Genentech: Employment. Tallman:Orsenix: Other: Advisory board; Cellerant: Research Funding; AROG: Research Funding; AbbVie: Research Funding; BioSight: Other: Advisory board; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding. Tiecke:Novartis: Employment. Pallaud:Novartis: Employment. de Witte:Amgen: Consultancy, Research Funding; Novartis: Research Funding; Celgene: Honoraria, Research Funding. Niederwieser:Novartis: Research Funding; Miltenyi: Speakers Bureau. Ehninger:Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees. Bullinger:Bristol-Myers Squibb: Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Pfizer: Speakers Bureau; Bayer Oncology: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Speakers Bureau; Sanofi: Research Funding, Speakers Bureau. Döhner:Pfizer: Research Funding; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; AROG Pharmaceuticals: Research Funding; Agios: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Pfizer: Research Funding. Thiede:Novartis: Honoraria, Research Funding; AgenDix: Other: Ownership.

Introduction: FLT3-mutations (FLT3mut) are the most common molecular alteration in younger adult patients with AML. The two major variants are internal tandem duplication (ITD) mutations mainly affecting the juxtamembrane domain and point mutations (mut) located in the second tyrosine kinase domain (TKD). FLT3-ITD mut, especially those with a high allelic ratio (AR), are associated with a high rate of treatment failure. In line with this, the 2017 European Leukemia Network (ELN) recommendations categorize patients with a high AR (i.e. >0.5 mutant/wildtype; wt) with NPM1-wt within the adverse risk group. The recent approval of midostaurin for the treatment of newly diagnosed FLT3-mutant AML underscores the need for reliable detection of FLT3mut. Several methods have been described, but most laboratories (labs) use PCR combined with capillary electrophoresis (CE), either alone (for ITD-mutations) or combined with a restriction enzyme digest (for TKD- point mutations). Despite the high clinical importance of FLT3mut, data on the diagnostic accuracy of available methods are scarce. We here describe our experience in FLT3mut testing gained in the context of the prospective international CALGB 10603/RATIFY study. Methods: CALGB 10603/RATIFY was a phase 3, randomized study in patients (18-0.7). Testing was performed in 9 reference labs across 6 countries using a harmonized PCR-method based on CE-detection (Stone et al, NEJM, 2017). PCR was done in triplicate starting from the DNA, mean values of these measurements were reported. To ensure consistency between labs, a cross validation quality control (CVQC) procedure was performed every 6 months. A set of 50 test samples (whole-genome amplified DNA from patients with known FLT3status) was prepared centrally and distributed in a blinded fashion. Per round, 25 of these samples (5 ITD wt, 5 ITD AR cut-off positive ≈0.05, 5 ITD AR ≈0.7, 5 TKD wt, and 5 TKD AR cut-off positive ≈0.05)were analyzed. Each lab measured the AR of each sample with 3 reactions. For the analysis reported here, we re-evaluated all data collected in this CVQC effort to study the overall accuracy of results as well as the intra- and interlab variability. Results:In 8 rounds of CVQC, 5700 single tests were performed between 12/2007 and 6/2011. Results were centrally scored using the criteria mentioned above. The overall consistency with respect to the qualitative information (FLT3mutpos./neg.) was high, with more than 95% of measurements meeting the prespecified criteria. We calculated the "true" value for each sample using the median for each sample over all performed analyses. The median "true" AR values were 0.72 (range 0.61-0.93; ITD-high), 0.06 (range 0-0.09; ITD-cut-off) and 0.07 (0.04-0.12; TKD-cut-off). Comparing these true values with the individual results, we saw considerable intra- and interlab variability with respect to accuracy of the measurements. The median error (ME) from this true value was 11.2% (95%CI 10.5-11.9%), it was lower for ITD high samples (7.1%; 95%CI 6.1-8.3%) than ITD-cut-off (13.4%; 95%CI 11.5-16.7%) and TKD (16.2%; 95%CI 13.3.-17.6%). The ME was significantly reduced, when the triplicate values were used, e.g. ME ITD AR 0.7 5.7% (95%CI 4.8-6.6%; p Conclusions:This first report of round robin testing for FLT3mutshows that using a standardized protocol, the qualitative assessment of FLT3mutis feasible with high accuracy. However, the assessment of the FLT3-AR clearly shows a considerable variability, which can be reduced by using a triplicate analysis, but still has to be taken into account, especially in patients evaluated for allogeneic stem cell transplantation. Further standardization of FLT3-testing appears highly warranted. Disclosures Thiede: AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding. Wei:Pfizer: Honoraria, Other: Advisory committee; Novartis: Honoraria, Other: Advisory committee, Research Funding, Speakers Bureau; Amgen: Honoraria, Other: Advisory committee, Research Funding; Celgene: Honoraria, Other: Advisory committee, Research Funding; Abbvie: Honoraria, Other: Advisory board, Research Funding, Speakers Bureau; Servier: Consultancy, Honoraria, Other: Advisory committee, Research Funding. Li:Novartis: Employment. Pallaud:Novartis: Employment. Tiecke:Novartis: Employment. Larson:BristolMyers Squibb: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Ariad/Takeda: Consultancy, Research Funding. Döhner:Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celator: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Pfizer: Research Funding; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Pfizer: Research Funding. Ehninger:GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Bayer: Research Funding; Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership.

Summary Background Patients with advanced basal cell carcinoma have limited treatment options. Hedgehog pathway signalling is aberrantly activated in around 95% of tumours. We assessed the antitumour activity of sonidegib, a Hedgehog signalling inhibitor, in patients with advanced basal cell carcinoma. Methods BOLT is an ongoing multicentre, randomised, double-blind, phase 2 trial. Eligible patients had locally advanced basal cell carcinoma not amenable to curative surgery or radiation or metastatic basal cell carcinoma. Patients were randomised via an automated system in a 1:2 ratio to receive 200 mg or 800 mg oral sonidegib daily, stratified by disease, histological subtype, and geographical region. The primary endpoint was the proportion of patients who achieved an objective response, assessed in the primary efficacy analysis population (patients with fully assessable locally advanced disease and all those with metastatic disease) with data collected up to 6 months after randomisation of the last patient. This trial is registered with ClinicalTrials.gov, number NCT01327053. Findings Between July 20, 2011, and Jan 10, 2013, we enrolled 230 patients, 79 in the 200 mg sonidegib group, and 151 in the 800 mg sonidegib group. Median follow-up was 13·9 months (IQR 10·1–17·3). In the primary efficacy analysis population, 20 (36%, 95% CI 24–50) of 55 patients receiving 200 mg sonidegib and 39 (34%, 25–43) of 116 receiving 800 mg sonidegib achieved an objective response. In the 200 mg sonidegib group, 18 (43%, 95% CI 28–59) patients who achieved an objective response, as assessed by central review, were noted among the 42 with locally advanced basal cell carcinoma and two (15%, 2–45) among the 13 with metastatic disease. In the 800 mg group, 35 (38%, 95% CI 28–48) of 93 patients with locally advanced disease had an objective response, as assessed by central review, as did four (17%, 5–39) of 23 with metastatic disease. Fewer adverse events leading to dose interruptions or reductions (25 [32%] of 79 patients vs 90 [60%] of 150) or treatment discontinuation (17 [22%] vs 54 [36%]) occurred in patients in the 200 mg group than in the 800 mg group. The most common grade 3–4 adverse events were raised creatine kinase (five [6%] in the 200 mg group vs 19 [13%] in the 800 mg group) and lipase concentration (four [5%] vs eight [5%]). Serious adverse events occurred in 11 (14%) of 79 patients in the 200 mg group and 45 (30%) of 150 patients in the 800 mg group. Interpretation The benefit-to-risk profile of 200 mg sonidegib might offer a new treatment option for patients with advanced basal cell carcinoma, a population that is difficult to treat. Funding Novartis Pharmaceuticals Corporation.

Angiogenesis, the formation of blood vessels, is a complex process involving numerous pathways and receptors that are essential for tumor growth and vascular metastasis. Tumors release a spectrum of proangiogenic cytokines, driven by metabolic and acidic environmental effects and hypoxia. Of them vascular endothelial growth factors (VEGFs) are essential regulators of tumor angiogenesis (Fig. 24.1) (Hicklin and Ellis 2005). The VEGF family consists of VEGF-A to VEGF-E and placental growth factors 1 and 2 which bind to three structurally related receptor tyrosine kinases, VEGFR-1, VEGFR-2, and VEGFR-3 (Fig. 24.2) (Takahashi and Shibuya 2005).

Background: Immunotherapy with monoclonal antibodies (mAbs) has recently entered the clinical arena in multiple myeloma, including Daratumumab that targets CD38 on malignant plasma cells. The efficacy of mAbs depends on antigen density and expression of accessory ligands on target cells to initiate cell- and complement-dependent effector mechanisms. Here, we investigate the use of the histone deacetylase inhibitor (HDACi) Panobinostat to modulate target antigen expression and ligand profile on myeloma in favor of potent mAb-mediated recognition and destruction. We show that Panobinostat augments CD38 expression specifically on myeloma cells and demonstrate powerful synergy with anti-CD38 mAb Daratumumab in pre-clinical models. Methods: The myeloma cell line MM1.S and primary myeloma cells were treated with titrated doses of Panobinostat (0, 10, 25 nM) and expression of CD38 and a panel of additional target molecules including B-cell maturation antigen (BCMA) and SLAMF7, as well as accessory ligands analyzed by flow cytometry at 24, 48 and 72 hours. Antibody-dependent cellular cytotoxicity (ADCC) against Panobinostat treated and untreated myeloma cells was analyzed at 4 and 20 hours after addition of PBMC at an effector to target ratio of 25:1 in the presence of Daratumumab (1, 10, 50 ug/mL) or an isotype control antibody. Results: We first treated the myeloma cell line MM1.S with Panobinostat and analyzed its direct cytotoxic anti-myeloma effect. Consistent with previous work, the percentage of live MM1.S myeloma cells had decreased to 85% and 50% after 48 hours of exposure to 10 and 25 nM respectively. We analyzed expression of CD38 on residual live, i.e. 7-AAD negative MM1.S cells by flow cytometry and observed a 1.5 (10 nM) and 2-fold (25 nM) increase of CD38 expression by mean fluorescence intensity (MFI) compared to baseline levels and untreated control cells. The increase in CD38 expression was already detectable after 24 hours and plateaued between 48 and 72 hours. We confirmed our observation in primary myeloma cells from multiple donors (n=4) and detected an even stronger increase to 2 (10 nM) and 4-fold (25 nM) higher CD38 expression compared to untreated cells at 48 hours. Interestingly, expression of BCMA and SLAMF7 was not increased after Panobinostat treatment at all tested concentrations and time points in both MM1.S and primary myeloma. We confirmed that Panobinostat-induced upregulation of CD38 specifically occurred in myeloma, and neither observed this phenomenon in a panel of leukemia and lymphoma cell lines including Raji (Burkitt) and JeKo-1 (mantle cell), nor on resting/activated primary CD8+ and CD4+ T cells that we isolated from peripheral blood of several donors (n=3). Next, we were interested in determining whether the increase in CD38 expression enabled superior anti-myeloma activity of the anti-CD38 mAb Daratumumab. Panobinostat pre-treatment was done for 48 hours at 10 nM as this is a clinically achievable serum level with currently approved regimens. Indeed, significantly higher ADCC was mediated by Daratumumab at all tested concentrations (1, 10 and 50 ug/mL) against MM1.S that we had exposed to Panobinostat. At 4 hours, ADCC was 45% and 25% in Panobinostat-treated and untreated MM1.S respectively, and at 20 hours, near-complete, >90% ADCC of Panobinostat-pre-treated MM1.S had occurred, whereas only 65% of MM1.S were eliminated by Daratumumab without Panobinostat pre-treatment. These data were confirmed in multiple experiments with MM1.S and PBMC from different donors, and with primary myeloma cells. Experiments to evaluate synergy of Panobinostat and Daratumumab therapy in a xenograft model (NSG/MM1.S) are ongoing. Conclusions: Our data demonstrate that the HDACi Panobinostat induces upregulation of CD38 on myeloma and a subsequent dramatic increase of Daratumumab-mediated ADCC in pre-clinical models. These data suggest that Panobinostat could be used synergistically with Daratumumab in a clinical setting to increase response rates and extend duration of responses to Daratumumab. Panobinostat has a known ability to modulate the transcriptional profile of myeloma cells and our data demonstrate for the first time that this ability can be utilized to augment the therapeutic index of antibody-based immunotherapy in multiple myeloma. Disclosures Pallaud: Novartis: Employment. Lehmann:Novartis: Employment. Hudecek:Novartis: Research Funding.

Background The hedgehog pathway inhibitor sonidegib demonstrated meaningful tumor shrinkage in more than 90% of patients with locally advanced basal cell carcinoma (BCC) or metastatic BCC in the BCC Outcomes with LDE225 Treatment study. Objective This report provides long-term follow-up data collected up to 12 months after the last patient was randomized. Methods In this multicenter, randomized, double-blind phase II study, patients were randomized 1:2 to sonidegib 200 or 800 mg. The primary end point was objective response rate assessed by central review. Results Objective response rates in the 200- and 800-mg arms were 57.6% and 43.8% in locally advanced BCC and 7.7% and 17.4% in metastatic BCC, respectively. Among the 94 patients with locally advanced BCC who responded, only 18 progressed or died and more than 50% had responses lasting longer than 6 months. In addition, 4 of 5 responders with metastatic BCC maintained an objective response. Grade 3/4 adverse events and those leading to discontinuation were less frequent with sonidegib 200 versus 800 mg (38.0% vs 59.3%; 27.8% vs 37.3%, respectively). Limitations No placebo or comparator arms were used because sonidegib demonstrated efficacy in advanced BCC in a phase I study, and the hedgehog pathway inhibitor vismodegib was not yet approved. Conclusion With longer follow-up, sonidegib demonstrated sustained tumor responses in patients with advanced BCC.

Background AVEREL efficacy and safety results have been reported. We present exploratory analyses from the optional BM substudy. Methods Patients (pts) with HER2-positive LR/mBC received 1st-line T (100 mg/m2 q3w) + H (8 → 6 mg/kg q3w) ± BV (15 mg/kg q3w). The primary endpoint was investigator-assessed PFS. Baseline (BL) blood and tissue samples were collected from consenting pts. BL plasma levels of candidate BMs were measured using a novel ELISA. Median BL values for each BM were used to define high (> median; H) vs low (≤ median; L) BM cohorts and to correlate BMs with PFS using Cox regression corrected for prognostic factors. Results Plasma samples were available from 162/424 pts (38%). ICAM-1 correlated statistically significantly with PFS at α = 0.05. Table: 1657P TH BV + TH Subgroup Events/pts, n Median, mo HR (95% CI) Events/pts, n Median, mo Interaction p value a All 64/82 11.2 66/80 16.5 0.78 (0.56–1.11) E-selectin L H 31/44 32/37 16.4 8.2 30/36 35/43 16.5 16.6 1.01 (0.61–1.68) 0.57 (0.35–0.92) 0.241 IL-8 L H 35/45 28/36 13.6 8.4 28/35 37/44 16.4 17.8 0.84 (0.51–1.40) 0.74 (0.45–1.22) 0.506 ICAM-1 L H 30/45 33/36 16.4 10.3 29/35 36/44 16.5 16.2 1.13 (0.68–1.89) 0.49 (0.30–0.80) 0.017 bFGF L H 32/41 31/40 13.3 11.1 33/39 32/40 16.4 19.1 0.80 (0.49–1.30) 0.80 (0.49–1.32) 0.837 PDGF-C L H 29/40 35/42 17.1 8.5 32/41 34/39 16.5 16.6 0.87 (0.52–1.44) 0.72 (0.45–1.16) 0.342 VEGF-A L 33/45 13.6 30/36 16.5 0.83 (0.50–1.36) 0.795 H 31/37 8.5 35/43 16.6 0.70 (0.43–1.14) VEGF-C L H 35/42 29/40 13.5 9.6 32/39 34/41 14.8 19.1 0.70 (0.43–1.15) 0.92 (0.56–1.51) 0.649 VEGFR-1 L H 30/40 33/41 11.1 13.3 33/40 32/39 16.2 16.6 0.76 (0.46–1.25) 0.84 (0.52–1.37) 0.982 VEGFR-2 L H 35/44 28/37 11.2 11.0 30/36 35/43 13.7 19.2 0.95 (0.58–1.55) 0.67 (0.40–1.10) 0.174 VEGFR-3 L H 35/48 28/33 12.2 11.0 25/32 40/47 15.3 16.6 0.88 (0.52–1.47) 0.65 (0.40–1.06) 0.051 a Model includes prognostic factors. Conclusions High BL ICAM-1 correlated significantly with greater PFS benefit from BV at α = 0.05, consistent with data in lung cancer. VEGF-A, VEGFR-2 and -3 and E-selectin showed potential predictive value. VEGF-A and VEGFR-2 results are in line with data in HER2-negative mBC and pancreatic cancer. Disclosure L. Gianni: LG acts as a Consultant and has sat on Advisory Boards for Roche, Genentech, GSK, Novartis, Pfizer, Boehringer Ingelheim, Astra Zeneca, and Celgene. A. Chan: AC has acted as a Consultant, sat on Advisory Boards, and received honoraria and research funding from Roche. X. Pivot: XP has acted as a Consultant and sat on Advisory Boards for Roche, Eisai and GlaxoSmithKline, and has received honoraria from Sanofi. R. Greil: RG has acted as a Consultant for, sat on Advisory Boards for, and received honoraria and research funding from Roche. L. Provencher: LP has received a travel grant and honoraria from Roche. S. Prot: SP is an employee of F Hoffmann-La Roche Ltd. N. Moore: NM is an employee and holds stock in F Hoffmann-La Roche Ltd. S.J. Scherer: SS is an employee of Genentech. C. Pallaud: CP is an employee of F Hoffmann-La Roche Ltd. . All other authors have declared no conflicts of interest.

Background GOG-0218 showed significantly improved progression-free survival (PFS) in patients (pts) receiving BV 15 mg/kg q3w concurrently with CT and continued alone until progression or for up to 15 mo. GOG-0218 includes extensive BM evaluation to identify pts benefitting most from BV. Analysis of plasma VEGF-A and VEGFR-2 was prioritised based on encouraging findings in BV trials in several tumour types. Table: 198P PFS OS Median, mo HR Median, mo HR Subgroup PL BV PL BV VEGF-A ≤ median 12.3 18.6 0.52 43.0 48.5 0.87 > median 11.0 17.5 0.70 33.9 41.8 0.78 ≤ Q3 12.3 18.6 0.59 45.1 48.5 0.89 > Q3 9.7 13.8 0.67 28.6 37.7 0.72 VEGFR-2 ≤ median 12.0 18.0 0.68 35.1 40.6 0.90 > median 10.4 18.2 0.53 38.4 48.5 0.69 ≤ Q3 11.0 17.3 0.63 38.2 41.8 0.87 > Q3 12.5 22.1 0.46 38.6 - 0.59 Q = quartile Methods Pts with newly diagnosed stage IV or macroscopic optimal stage III OC were randomised to 6 cycles (c) of CT with: placebo (PL) c2-22 (arm A); BV c2-6 → PL c7-22 (arm B); or BV c2-22 (arm C). Post-surgery pre-CT plasma samples were analysed using a multiplex ELISA. Baseline (BL) BM levels were used to dichotomise pts. Potential interactions between BM levels and PFS (1° endpoint) and overall survival (OS; 2° endpoint) were tested using log-rank testing and Cox regression approaches. Results Post-surgery samples were available from 582 of 1248 pts in arms A and C. Median BL VEGF-A and VEGFR-2 levels were 144.3 pg/mL and 14.7 ng/mL, respectively. No significant interaction was seen at α = 0.05. Exploratory analyses with other cut-offs are hypothesis generating for potential predictive (VEGF-A and VEGFR-2) or prognostic (VEGF-A: OS) value. Exploratory analyses revealed no correlation between plasma VEGF-A and time since surgery. Conclusions The potential prognostic (VEGF-A) and predictive (VEGF-A, VEGFR-2) value seen in breast, pancreatic and gastric cancers was not apparent in post-surgery samples from GOG-0218 using a median cut-off. Results with other cut-offs provide a rationale for further investigation of potential prognostic and predictive value. Findings may reflect differing biology and interplay between VEGF-A isoforms across tumour types. The possible impact of pre- vs post-surgery samples is also being investigated. Disclosure R.A. Burger: RB has served on Advisory Boards for Roche/Genentech. R. Mannel: RM has served on Advisory Boards for Genentech. V. Henschel: VH is employed by and holds shares in Roche. M. Sovak: MS is an employee of Genentech. S.J. Scherer: SS is an employee of Genentech Inc. S. De Haas: SdH is an empoloyee of F Hoffmann-La Roche Ltd. C. Pallaud: CP is an employee of F Hoffmann-La Roche Ltd. All other authors have declared no conflicts of interest.

WCN 2013 No: 1207 Topic: 36 — Other topic Efficacy and biomarker findings from AVaglio, a phase III trial of bevacizumab plus temozolomide and radiotherapy in newly diagnosed glioblastoma W. Wick, T. Cloughesy, W. Mason, R. Henriksson, F. Saran, R. Nishikawa, M. Hilton, J. Garcia, C. Pallaud, O. Chinot. University Medical Centre, Heidelberg, Germany; University of California, Los Angeles, CA, USA; Princess Margaret Hospital, University of Toronto, Toronto, ON, Canada; Regional Cancer Center Stockholm, Stockholm, Sweden; Umea University, Umea, Sweden; The Royal Marsden NHS Foundation Trust, Surrey, UK; International Medical Center, Saitama Medical University, Saitama, Japan; F. Hoffmann-La Roche, Basel, Switzerland; Aix-Marseille University, AP-HM, Service de Neuro-Oncologie, CHU Timone, Marseille, France Background: Glioblastoma has a high disease burden and poor prognosis. AVAgliowas the first double-blind, placebo-controlled phase III study evaluating bevacizumab in newly diagnosed glioblastoma. Objectives: To evaluate efficacy, safety and potential biomarkers (plasma VEGF-A/VEGFR-2 prioritised). Patients and methods: Patients received single-agent bevacizumab or placebo plus standard-of-care treatment (radiotherapy plus temozolomide) until progression/unacceptable toxicity. Co-primary endpoints were investigator-assessed PFS and OS. Secondary endpoints included health-related quality of life (HRQoL). Exploratory endpoints included correlative biomarker analysis, KPS, corticosteroid use. Results: Baseline characteristics were balanced for intent-to-treat (n = 921) and biomarker-evaluable (n = 571) populations. Bevacizumab significantly prolonged PFS (HR = 0.64, 95% CI 0.55– 0.74, p b 0.0001; median 10.6 vs 6.2 months) and delayed HRQoL time to definitive deterioration (p b 0.0001). Interim OS did not cross the threshold for significance (HR = 0.89, 95% CI 0.75–1.07, p = 0.2135). Functional independence (KPS ≥ 70%) was maintained during PFS in both arms (median bevacizumab vs placebo: 9 vs 6 months). Patients treated with bevacizumab had a diminished corticosteroid requirement. Patients with low and high baseline plasma VEGF-A concentrations derived similar PFS benefit with bevacizumab (p = 0.610): low VEGF-A (HR = 0.64, 95% CI 0.48– 0.84) versus high (HR = 0.59, 95% CI 0.45–0.78). Similarly, PFS benefit in patients with low and high baseline VEGFR-2 levels was comparable (p = 0.736): low VEGFR-2 (HR = 0.54, 95% CI 0.41– 0.71) versus high (HR = 0.66, 95% CI 0.50–0.87). Conclusion: Addition of bevacizumab to standard-of-care therapy provided a significant clinically meaningful PFS improvement with stable/improved HRQoL/KPS and reduced corticosteroid requirement. NoVEGF-A/VEGFR-2 predictive/prognostic effectwas observed. Interim OS analysis did not cross the threshold for significance. doi:10.1016/j.jns.2013.07.2268 Abstract — WCN 2013 No: 2103 Topic: 36 — Other topic Flow cytometric analysis of cerebrospinal fluid is low diagnostic yield without atypical morphology or prior history of hematologic malignancy WCN 2013 No: 2103 Topic: 36 — Other topic Flow cytometric analysis of cerebrospinal fluid is low diagnostic yield without atypical morphology or prior history of hematologic malignancy G.H.J. Stevens, A.M.B. Collie, B.T. Hill, K. Fenner, E. Gazdick, L.A. Rybicki, E.H. Hsi. Rose Ella Burkhardt Brain Tumor and Neuro-Oncology Center, Cleveland Clinic, Cleveland, OH, USA; Pathology, Cleveland Clinic, Cleveland, OH, USA; Taussig Cancer Center, Cleveland Clinic, Cleveland, OH, USA; Quality Health Science, Cleveland Clinic, Cleveland, OH, USA Background: Flow cytometric analysis (FCA) of cerebrospinal fluid (CSF) has utility in detecting central nervous system (CNS) involvement by hematologic malignancies. Yet, the majority of samples are negative by FCA. Objective: Identify pre-test characteristics of CSF specimens that will allow the rational use of FCA in the diagnosis of hematologic malignancy in CSF. Design: Retrospective data was collected from the medical record and flow cytometric reports for all CSF samples submitted for FCA between 2007 and 2009. Patients: 423 patients for whom 501 CSF samples were submitted for FCA. Results: A positive diagnosis of a hematologic malignancy was made in 41 specimens (8.2%). The FCA-positive specimens showed atypical morphology, either blasts or atypical lymphocytes, in 98% of Abstracts / Journal of the Neurological Sciences e629 (2013) e629–e678 e654

2023^ Background: Bv plus 1st-line standard of care (SoC; T+RT) achieved a PFS benefit in the AVAglio phase III, randomized, double-blind, placebo [P]-controlled trial in patients (pts) with newly diagnosed GBM. AVAglio includes BM evaluation to identify pts benefiting most from Bv. Analysis of plasma VEGF-A and VEGFR-2 was prioritized based on encouraging findings in Bv trials in several tumor types. Methods: AVAglio includes an optional, exploratory correlative BM analysis; participating pts provided informed consent for BMs. Baseline (BL) plasma samples were analyzed using the Roche IMPACT platform, based on multiplex ELISA technology. Pts were dichotomized according to BM levels using either Q1, median or Q3 cut-offs. Potential interactions between BL BM levels and PFS were tested using Cox regression analyses. Results: Of 921 patients enrolled, 571 (62%) were evaluable in the BM study. Baseline characteristics and PFS outcome were comparable in the ITT and BM-evaluable populations. Median BL VEGF-A and VEGFR-2 levels were 77.0 pg/mL and 12.6 ng/mL, respectively. No significant interaction for PFS was seen at α=0.025. Conclusions: The potential predictive (VEGF-A, VEGFR-2) and prognostic (VEGF-A) value seen in breast, pancreatic and gastric cancers was not apparent in BL BM samples from AVAglio using a median, Q1 or Q3 cut-off. Additional plasma and tumor BM analyses are ongoing. Clinical trial information: NCT00943826. [Table: see text]

Background BO21015 (NCT00700180) is a phase II, randomized, multicentre study exploring correlation between biomarkers (BMs) and best overall response (BOR) to bevacizumab with carboplatin/gemcitabine (CG) or carboplatin/paclitaxel (CP) in chemonaive patients with advanced/recurrent NSCLC. Efficacy, safety and correlation of 7 baseline (BL) plasma BM (bFGF, E-selectin, ICAM, PLGF, VEGFA, VEGFR-1 and VEGFR-2) with BOR and progression-free survival (PFS) have been reported.1 This abstract presents BM analysis for tumour tissue, plasma time course and clinical outcome. Methods 303 eligible patients were randomized 1:1 to receive bevacizumab 7.5 mg/kg or 15 mg/kg until disease progression (PD) or unacceptable toxicity (with 6 cycles of CG or CP, at investigators' discretion). Consented patients provided blood1 and tumour samples for BM analysis. Pre-specified exploratory analyses examined correlation between BL plasma BM and overall survival (OS) and changes in plasma BM levels from BL to PD, cycles 2, 4 and 6. Plasma BM levels were measured by ELISA. IHC analyses of 5 tumour BMs (VEGFR-1, MVD, VEGFA, VEGFR-2 and NRP1) were assessed for correlation with BOR, PFS and OS, and with BL plasma BM levels. Results \Further exploratory analyses adjusting for BL prognostic factors and accounting for multiple testing showed a correlation of high BL VEGFA levels (3median) with shorter OS (n = 280; 19.8 vs 11.1 mos; p = 0.0042). No other BL plasma BMs correlated with OS. No significant changes in plasma BM levels were seen between baseline and PD, and/or cycles 2, 4 or 6 for any of the BMs. The only correlation between tumour and plasma markers was for tumour VEGFR1 expression and VEGFA plasma BL (p = 0.025, 0.26). No significant correlation was seen between tumour BM level and BOR, PFS or OS. Conclusions Exploratory analysis showed high plasma BL VEGFA significantly correlated with shorter OS, consistent with previously reported data on PFS. No other BL plasma BMs correlated with OS. BL plasma VEGFA levels correlated with tumour VEGFR1 expression. None of the investigated tumour BMs significantly correlated with clinical outcome. 1 Mok et al. ESMO 2011 Disclosure M. Reck: Attended advisory boards for Roche, Lilly, BMS, AstraZeneca and Daiichi Sankyo. Received honoraria for lectures from Roche, Lilly, Daiichi Sankyo and AstraZeneca. V.A. Gorbunova: Attended advisory boards with Novartis and Pfizer. Honoraria for lectures from Roche and Bayer. B. Szima: Received funding for research. C. Yu: Attended advisory boards for Roche, AstraZeneca, Pfizer and Takeda. C. Pallaud: Owns stock in Roche. Currently employed by Roche. S.J. Scherer: Currrently employed by Roche/Genentech. V. Archer: Currently employed by Roche. T.S.K. Mok: Advisory boards: AstraZeneca, Roche, Eli Lilly, Merck Serono, Eisai, BMS, BeiGene, AVEO, Pfizer, Taiho, BI, GSK Biologicals. On the IASLC board of directors. Received research funding from AstraZeneca. Employed by The Chinese Uinversity of Hong Kong. All other authors have declared no conflicts of interest.

Background ABIGAIL (BO21015; NCT00700180) is a phase II, randomized, multicentre study exploring correlation between biomarkers (BMs) and best overall response (BOR) to bevacizumab with carboplatin/gemcitabine (CG) or carboplatin/paclitaxel (CP) in chemonaive patients with advanced/recurrent ns-NSCLC. ABIGAIL efficacy, safety and plasma baseline results have been reported. This abstract presents exploratory clinical genotyping data from this study. Methods 303 patients with untreated advanced/recurrent ns-NSCLC were randomized 1:1 to receive bevacizumab 7.5 mg/kg or 15 mg/kg until progression or unacceptable toxicity (with 6 cycles of CG or CP). Patients who consented provided blood and tumour samples for BM analysis. Exploratory analyses were conducted to assess whether genetic variants in the VEGFA pathway may act as biomarkers for efficacy and safety. Here we report data from DNA analysis for 12 single-nucleotide polymorphisms (SNPs) across 3 genes: VEGFA (5 SNPs), VEGFR-1 (3 SNPs) and VEGFR-2 (4 SNPs). SNPs were identified using specific individual genotyping assays. Results VEGFA: c. + 405/c.-634 (CG), VEGFA: c.-460T > C; c. -1498T > C (CT) and VEGFA: c.-2578 C > A (AC) were all associated with >50% higher odds of responding to treatment. VEGFR-1 rs9554316 (GT) was associated with >30% higher risk of progression and >40% higher risk of death. VEGF: c. + 936 C > T (CT) was associated with higher incidence of hypertension. When p-values were adjusted for treatment and prognostic factors, no SNPs were associated with significantly higher risk of hypertension. Conclusions One SNP was associated with increased risk of progression/death, while 3 others were associated with increased BOR. However, adjustment for multiple testing would no longer result in statistically significant p-values. SNPs analysed in this study have been previously reported as showing potential predictive value in other studies: VEGFA SNPs in breast cancer (E2100) and NSCLC (E4599); VEGFR1 SNP in pancreatic cancer (AVITA). More exploratory analyses from this and other trials of bevacizumab may provide further insight. Disclosure C. Pallaud: Own stock in and currently employed by F. Hoffmann-La Roche Ltd. M. Reck: Attended advisory boards for Roche, Lilly, BMS, AstraZeneca and Daiichi Sankyo. Received honoraria for lectures from Roche, Lilly, Daiichi Sankyo and AstraZeneca. B. Szima: Received funding for research. C. Yu: Attended advisory boards for Roche, AstraZeneca, Takeda and Pfizer. S.J. Scherer: Currently employed by Roche/Genentech. V. Archer: Currently employed by Roche. T.S.K. Mok: Attended advisory boards for AstraZeneca, Roche, Eli Lilly, Merck Serono, Eisai, BMS, BeiGene, AVEO, Pfizer, Taiho, BI and GSK Biologicals. On the IASLC board of directors. Received research funding from AstraZeneca. All other authors have declared no conflicts of interest.

Introduction: Avastin Biomarkers In lunG And 3D Innovative anaLysis (ABIGAIL), which is a phase II, open-label, randomized study, investigated correlations between biomarkers and best overall response to bevacizumab plus platinum-doublet chemotherapy for patients with advanced/recurrent non–small-cell lung cancer. Methods: Patients received bevacizumab (7.5 or 15 mg/kg, 3-weekly until disease progression/unacceptable toxicity) plus carboplatin/gemcitabine or carboplatin/paclitaxel (maximum six cycles). Plasma samples (baseline/throughout treatment) were analyzed for vascular endothelial growth factor (VEGF)-A (baseline only), VEGF receptors (VEGFR-1/VEGFR-2), basic fibroblast growth factor, E-selectin, intercellular adhesion molecule-1, and placental growth factor (baseline only). Tumor samples (primary specimen) were analyzed for VEGF-A, VEGFR-1/VEGFR-2, neuropilin (NRP), and CD31. Response was evaluated at baseline and every 6 weeks (Response Evaluation Criteria in Solid Tumors). Results: Patients were randomized to receive chemotherapy plus 7.5 mg/kg ( n = 154) or 15 mg/kg ( n = 149) bevacizumab. For the primary analysis, none of the baseline plasma biomarkers correlated with best overall response. Exploratory analyses showed that low VEGF-A levels were associated with longer progression-free survival (7.4 versus 6.1 months; hazard ratio, 1.57; 95% confidence intervals, 1.17 to 2.09; p = 0.002) and overall survival (19.8 versus 11.1 months; hazard ratio, 1.57; 95% confidence interval, 1.15–2.13; p = 0.004) compared with these in high baseline plasma VEGF-A levels. No plasma biomarkers changed significantly over time. No significant correlations were observed between tumor biomarkers and clinical outcomes. No new safety signals were observed. Conclusion: Baseline and/or dynamic changes in plasma basic fibroblast growth factor, E-selectin, intercellular adhesion molecule-1, placental growth factor, VEGFR-1 and VEGFR-2, and tumor biomarkers did not correlate statistically with treatment outcomes for bevacizumab plus chemotherapy. Only baseline plasma VEGF-A was significantly correlated with progression-free survival/overall survival.

BACKGROUND: Cytotoxic chemotherapy/agents can cause a range of side effects in cancer patients including anemia, neutropenia and thrombocytopenia, resulting in increased morbidity and mortality. These cytopenias are in part due to massive depletion of bone marrow progenitors. A common target of many chemotherapies is the megakaryocyte (MK), a rare progenitor representing 0.2% of the bone marrow. Loss of MK lineage cells results in thrombocytopenia, therefore, discovering ways to protect MKs from cytotoxic drugs could prevent this life-threatening condition. Here, we show that eltrombopag (EP), a small molecule, non-peptide thrombopoietin receptor (TPO-R) agonist, protects healthy MKs from different cytotoxic agents. We further explored the impact of cytotoxic drugs and EP treatment on signaling molecules active in MKs. METHODS: Peripheral blood and bone marrow samples were collected from healthy donors after informed consent and following protocols in accordance with the Declaration of Helsinki. CD34+ positive cells were isolated by immuno-magnetic bead separation from the samples and expanded for 8-10 days with a cytokine cocktail to induce MK differentiation. After MK expansion, cells were transferred to 96-well plates pretreated with 14 different drugs including cytarabine, gemcitabine, paclitaxel, carboplatin, cisplatin, doxorubicin, vincristine, etoposide, midostaurin, ruxolitinib, panobinostat, azacitidine, venetoclax and navitoclax. The drugs were plated in 5 different doses in a 10,000-fold concentration range, and cells added with or without EP. After 3 days incubation, the cells were analyzed on a high throughput flow cytometer using Annexin V and 7AAD to distinguish live from dead cells. To understand the impact of the drugs and EP on signaling molecules downstream of TPO-R, we analyzed phosphorylation of AKT, ERK, STAT3 and STAT5 in populations defined by MK markers CD41a, CD42b and CD110 (TPO-R). RESULTS: We found that EP overall supports MK survival in the presence of cytotoxic agents. The greatest net effect was observed when EP was combined with BCL2 inhibitors (venetoclax, navitoclax), which resulted in increased MK cell maturation compared to inhibitor alone, while gemcitabine plus EP showed the next best response. EP combined with etoposide, vincristine, cytarabine, paclitaxel, cisplatin and ruxolitinib resulted in an increase of immature MKs without a reduction in mature MK cell numbers. Higher numbers of MKs were observed when EP was combined with midostaurin, carboplatin, panobinostat and doxorubicin, compared to treatment without EP; although these numbers were lower than other tested drugs. Phosphoflow analysis revealed intriguing differences in EP-induced signaling compared to signaling induced by recombinant human thrombopoietin (rhTPO) (Figure 1). Also, EP-induced signaling did not correlate with TPO-R expression. EP activated AKT in all MK subsets, but rhTPO induced AKT only in immature MKs. While rhTPO induced ERK phosphorylation in different MK subsets, EP had no effect on ERK activation. Furthermore, rhTPO activated STAT3 only in mature MKs, but EP induced STAT3 in all MK populations. In contrast, both EP and rhTPO induced phosphorylation of STAT5 in all MK subsets. Overall signaling was inhibited by a selected set of agents (venetoclax, etoposide, midostaurin) compared to basal signaling, but addition of EP prevented the inhibition. Similar to the cell survival analysis, venetoclax combined with EP resulted in complete recovery of signaling activity compared to venetoclax alone. But the response was not as striking for etoposide or midostaurin combination compared to treatment alone. CONCLUSION: Using MKs expanded from CD34+ cells, we found that EP supports MK survival in the presence of several different cytotoxic agents, with the most striking rescue observed when EP was combined with BCL2 inhibitors. Importantly, EP and rhTPO induced distinct signaling patterns, with EP signaling independent of TPO-R expression, suggesting alternate EP targets. The tested drugs inhibited basal signaling in MKs, but this inhibition was prevented by EP. These results suggest addition of EP to cancer treatment regimens may prevent the inhibitory effects of some agents on MKs and support future investigations to demonstrate clinical efficacy of EP in combinations with some of these cytotoxic agents to alleviate thrombocytopenia. Figure 1. Figure 1. Disclosures Javarappa: Novartis Pharma AG: Research Funding. Tsallos:Novartis: Research Funding. Marques Ramos:Novartis: Employment. Pallaud:Novartis: Employment. Heckman:Celgene: Research Funding; Novartis: Research Funding; Orion Pharma: Research Funding.