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ABL1 Kinase Domain (ABL1-KD) mutations are a common resistance mechanism to tyrosine-kinase inhibitors (TKIs) in Chronic Myeloid Leukemia (CML) and Philadelphia Positive Acute Lymphoblastic Leukemia (ALL). Different ABL1-KD mutations induce different degrees of resistance to different TKIs. The early detection of these resistant mutations helps to adjust patient's treatment. Here we present an Ultra-Deep Sequencing approach to detect and quantify acquired ABL1-KD mutations in genomic DNA (gDNA), aiming to define a robust test to detect such alterations in TKIs exposed Philadelphia-Positive Leukemia Patients with a resolution below 1E-4. Firstly, we defined an ABL1 specific next-generation sequencing (NGS) panel designed to cover all coding regions of ABL1 exons 4-10. The 9 amplicons were designed to cover full exons where possible to detect co-occurring mutations (Figure 1A). A panel was then applied to 3 biological replicates of 3 Healthy control donors (9 NGS data points each with 220ng of gDNA). The average coverage per amplicon in all samples was at least 500,000x. The NGS data was then analyzed applying the NGS-MRD algorithm described elsewhere (Onecha, E et al. Haematologica 2019) to 25 known ABL1-KD hotspots. After applying our error correcting algorithm, we obtained an average of 135,000 (22,000-503,000) refined reads for the 25 hotspots. The limit of detection (LOD) was calculated for every position in the DNA as the mean noise (Variant Read Frequency; VRF) per position in the controls ± 3SD (standard deviation); the limit of quantification (LOQ) being defined as mean ± 10SD. For all the hotspots analyzed, the LOD was below 1E-4 and the LOQ below 3E-4 (Figure 1B), except for p.F311L (c.931T>C; LOD=2.7E-3). The high level of noise in this position, constant in the different control samples sequenced in different sequencing runs, is most likely related to the high number of homopolymers in the region. Ten Philadelphia-Positive Leukemia patients were then screened after TKI treatment (8 CML and 2 ALL). The median BCR-ABL1 defined by quantitative PCR (ratio BCR-ABL1 vs ABL1) in these follow-up samples was 0.6% (0.034% - 95%). All patients were screened in triplicates (220ng gDNA each) and the data-points ± 1SD from the mean were considered outliers (NGS false positives) and excluded from further analysis. Five patients presented a signal above the LOD for p.T315I (c.944C>T). This position is covered by 2 different amplicons in our panel. By bioinformatically demultiplexing the signal, the detection of those five mutations in both amplicons was confirmed (Amp_4; LOD=3E-5, Amp_5; LOD=4E-5). Moreover, aiming to validate this new approach, we applied to paired RNA samples an in-house BCR-ABL1/ABL1 nested PCR + NGS approach designed to quantify those alterations in cDNA. This approach confirmed the presence of 4 out of 5 gDNA detected mutations, with a Pearson correlation of 0.92 (Pval Here we show an Ultra-Deep NGS based test which allows the early detection of TKI resistant emerging clones in genomic DNA samples with a resolution of 1E-4. Despite the facts that in Phi-positive leukemia patients' other techniques such as the nested PCR are available, for most of heme- dyscrasias it is not easy to detect acquired mutations below 1% VRF. Our test can reduce this limit by at least 2 logarithms. The clinical impact of this approach is illustrated by the two LLA patients included, both under dasatinib therapy when the p.T315I mutations were detected. Those 2 patients were changed to ponatinib, reducing BCR-ABL1 levels. An extension of the cohort and the validation of our test at clinical level will be presented at the meeting. Figure Disclosures Heredia: Altum sequencing: Current Employment. Carrillo:Altum sequencing: Current Employment. Rufian:Altum sequencing: Current Employment. Wang:Hosea Precision Medical Technology Co., Ltd: Current Employment. Ribera:Pfizer, Amgen: Research Funding; Pfizer, Amgen, Ariad, Novartis: Consultancy, Speakers Bureau. Martinez-López:Janssen, BMS, Sanofi, Novartis, Incyte, F. Hoffmann-La Roche and Amgen: Honoraria, Other: Advisory boards; Hosea and Altum: Membership on an entity's Board of Directors or advisory committees; Janssen, Novartis, BMS, Incyte: Consultancy.

Background: Ruxolitinib is the standard of care for myelofibrosis (MF). However not all patients respond to ruxolitinib and other lose response while on treatment or require discontinuation due to adverse events. Therefore, several clinical trials with ruxolitinib in combination with other drugs are being conducted. We previously showed the synergistic effect of the combination ruxolitinib/nilotinib/prednisone in ex vivo models of peripheral blood mononuclear cells from MF patients and cell lines. Aims: This phase Ib/II study, non-randomized, open-label, evaluates safety and tolerability of ruxolitinib in combination with nilotinib and prednisone in patients with naïve or ruxolitinib-resistant MF. Methods:Between November 2017 and June 2020, 21 patients were included in RUNIC (EudraCT Number:2016-005214-21) at 6 Spanish sites. Six patients were considered screening failures. A total of 15 patients received at least one dose of ruxolitinib and were included in the intention-to-treat (ITT) or the per-protocol (PP) populations. The study design is summarized in figure 1. Out of 15 evaluable patients whose median age was 66.5 years (53.4-70.8). Six patients (40%) had a diagnosis of primary MF, 4 patients (26.7%) post-polycythemia vera MF, and 4 patients (26.7%) post-essential thrombocythemia MF. Most patients had International Prognostic Scoring System (IPSS) intermediate-1 or -2 disease (5 [33.3%] and 4 [26.7%], respectively), and 9 patients (60%) had JAK2 V617F mutation. Most of the patients (n=13; 86.7%) had received ruxolitinib as previous anti-neoplastic therapy for MF. Results:Eight patients completed 7 cycles (53.3%), and six patients 12 cycles (40%) and nine patients withdrawn because of disease progression, lack of efficacy, or no clinical benefit (n=5), withdrawal of consent (n=2), adverse event (AE) (pulmonary thromboembolism, n=1), and investigator's medical judgment (n=1). All patients experienced at least one AE during the study (the most common were hyperglycemia, asthenia and thrombocytopenia, and 14 patients registered at least one treatment-related AE (the most common were hyperglycemia (22.2%), thrombocytopenia (13.3%), and anemia (8.9%)). Five treatment-related serious adverse events (SAEs) were reported in 2 patients (13.3%), all of them related to nilotinib: one patient had pericardial effusion and bilateral pleural effusion, the other had congestive heart failure, pleural effusion, and pulmonary hypertension. No deaths were registered throughout the study.There were 2 patients with at least one adverse event meeting criteria for dose-limiting toxicity: grade 4 anemia and lumbar pain. Only eight patients were evaluated for spleen lengths at screening and cycle 7 (Figure 2B). Of these, 6 experienced a palpable spleen reduction, 4 of them with a 100% reduction. In contrast, one patient remained with the same spleen length, and one patient experienced a 25% increase.Six patients had both symptom evaluation at screening and cycle 7 (Figure 2D). Four experienced a total symptom score reduction, one patient remained with the same total symptom score, and two patients reported an increase.Six patients had spleen length measures recorded both at screening and at cycle 12. From those, 4 showed a spleen length reduction, 3 of them with a 100% reduction. Conclusions: Ruxolitinib in combination with nilotinib and prednisone showed relevant clinical activity in patients with naïve or ruxolitinib-resistant MF. The tolerability of this combination was acceptable, and the hyperglycemia was the treatment-related AE most frequent. The main cause of trial withdrawn was disease progression, lack of efficacy, or no clinical benefit. Based on these results, this triple combination should be explored in phase II/III clinical trials. Figure 1 Figure 1. Disclosures Ayala: Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Celgene: Honoraria. Garcia Gutierrez: BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Osorio: Janssen, Abbvie, Roche: Consultancy. Sanchez: Janssen, Jazz Pharmaceuticals, Gilead, Novartis, Amgen: Consultancy. Martínez-López: Janssen, BMS, Novartis, Incyte, Roche, GSK, Pfizer: Consultancy; Roche, Novartis, Incyte, Astellas, BMS: Research Funding.

Background. Increasing age is a risk factor for vascular events but also for bleeding in immune thrombocytopenia (ITP). In elderly, meta-analysis of clinical trials of romiplostim (ROM) and eltrombopag (ELT) show that thrombopoietin receptor agonists (TPO-RA) are effective and safe with the exception of increased thromboembolic risk (Olney et al, 2011; Michel et al 2015). Objective. To analyze how age influences the selection of TPO-RA, bleeding and thrombotic risk, comorbidities, and therapeutic management of ITP patients in a real-world setting. Methods. We conducted a multicenter retrospective study that included 121 adult patients with primary ITP from 19 secondary and tertiary Spanish hospitals who had initiated long-term therapy with ROM or ELT between January 2012 and December 2014. Information was collected from medical records (November 2016 to January 2018) to assess variables related to patient characteristics and outcomes of elderly (> 65 years; n=54) compared with younger individuals (n=67). Results. Patients included initiated TPO-RA (ROM, n=54; ELT, n=67) and maintained this therapy for a median time to collection of data of 35.2 months (1 to 67.3 months). The median age at diagnosis of elderly and younger cohorts was respectively 75 years (66-96 years), and 48 years (19-65 years). Older age was associated with a previous history of vascular events (VE) (P=0.049), with more patients receiving antithrombotic therapy (P=0.001), and with a non-significant trend towards increased risk of current VE under TPO-RA therapy (Table). During treatment, 15 patients experienced 17 VE (9 arterial, 8 venous); no association was found between risk for VE in patients under TPO-RA and past history of thromboembolic or ischemic events (P=0.727). Patients that were offered TPO-RA at younger ages presented at diagnosis with significantly lower platelet counts, and increased cumulative bleeding score (Page et al, 2007) (P=0.003, and P=0.034) than elderly ones. Younger patients also had significantly higher visceral bleeding rates at the onset of TPO-RA therapy (P=0.042) and had increased requirement for hospital care (emergency treatment or hospital admission) both six months before and after the start of TPO-RA (P=0.016, and P=0.002, respectively). Older age was associated with comorbidities such as hypertension and diabetes (P65 versus ≤65 years, however a more conservative management in terms of discontinuation of therapies was confirmed. Therefore, the rate of tapering off TPO-RA was significantly lower in those >65 years (P=0.028), although the proportion of patients that achieved therapy free response (TFR) (platelet count >50x109/l for at least 6 months) upon discontinuation was similar in both groups (Table). Conclusion. The management of older patients with chronic ITP is still challenging, and widespread effort is made to avoid potential complications such as those related to splenectomy. Our study reflects that the introduction of TPO-RAs has caused a change in the outcomes of these patients. The increased awareness of the unfavorable conditions that are present in this population induces a preferential use of TPO-RAs in elderly patients with a lower bleeding history than in younger patients. Although these drugs associate with a potential risk of increased thrombotic risk, our data indicate that past thrombotic history does not predispose to the development of VE; rather neoplasia in elderly patients, and splenectomy at younger ages are factors that increase the likelihood to suffer from these events. The compromise towards effective therapies in these fragile patients associates with low discontinuation of TPO-RA to test for TFR, although once tapered off, sustained platelet responses are similar to those in younger patients. Disclosures Mingot-Castellano: Novonordisk: Consultancy; Sobi: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Bayer: Consultancy; CSL Behring: Consultancy; Roche: Consultancy; Novartis: Consultancy. Jarque:Abbie: Consultancy, Speakers Bureau; Alexion: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; CellTrion: Consultancy; Gilead: Consultancy, Speakers Bureau; Grifols: Consultancy; Janssen: Consultancy, Speakers Bureau; MSD: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Servier: Speakers Bureau; Shionogi: Consultancy, Speakers Bureau; Shire: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau. Campos:Novartis: Speakers Bureau; Amgen: Speakers Bureau. Lopez Fernandez:Amgen: Consultancy, Speakers Bureau. Valcarcel:MSD: Consultancy, Honoraria, Speakers Bureau; Astellas: Consultancy, Honoraria, Speakers Bureau; JAZZ: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau. Casado:Amgen: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Álvarez Roman:Takeda: Research Funding; Amgen: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Speakers Bureau; NovoNordisk: Consultancy, Speakers Bureau; Sobi: Consultancy, Speakers Bureau.

Background . In clinical practice, tapering off thrombopoietin receptor agonists (TPO-RA) in immune thrombocytopenia (ITP) is considered if therapy is no longer needed due to a decrease in the disease activity favoring sustained treatment-free responses (TFR). To date, there are no predictors to identify when this approach is likely to be successful, other than earlier start of TPO-RA, and robust platelet responses. Aim. To evaluate clinical predictors of TFR in a real-world cohort of ITP patients treated with TPO-RA by dealing with confounding variables that could be minimized at the stage of the analysis. Methods. In this retrospective, multicenter study from 19 secondary and tertiary Spanish hospitals, patients aged >18 years with chronic ITP who had initiated TPO-RA (eltrombopag [EPG] or romiplostim [ROM]) between January 2012 and December 2014 were included. Data were collected from medical records (November 2016 to January 2018) on patient characteristics, history of disease and previous therapies, TPO-RA administration, response and discontinuation. Results. A total of 82 patients with a median age of 63 years (range 19-90 years), 59.9% females initiated TPO-RA (ROM, n=37; EPG, n=45). The median time from diagnosis of ITP to therapy with TPO-RA was 5.5 years (1.1-50.3 years). In all cases the TPO-RA was started with intention to treat indefinitely; the median initial doses of EPG were 350 mg/week (175-525 mg/week), and those of ROM 2.0 μg/kg/week (1.0-7.0 μg/kg/week). The median time on TPO-RA treatment was 2.9 years (0.1 to 5.6 years), and the median follow-up from start of TPO-RA until collection of data was of 3.9 years (1.3 to 5.6 years). A total of 29 patients (35.4%) switched TPO-RA during follow-up. The most frequent cause for switching was lack of efficacy (44.8% of cases -in all cases the initial TPO-RA was EPG-). Due to switching, 58 patients received ROM, and 53 were treated with EPG, yielding 122.3, and 100.8 patient-years of total exposure, respectively. During follow-up almost one half of the patients (47.6%, n=39) stopped TPO-RA. After a median of 1.4 years (0.1-3.3 yrs) under TPO-RA treatment, 19 patients (23.2% of the whole cohort) maintained TFR defined as platelet counts >50x109/l for a median follow-up of 2.8 years (1.5-4.4 years) in the absence of any platelet increasing drug. Remarkably, while the specific TPO-RA that was discontinued did not influence the probability to achieve TFR (P=0.582), there was a trend towards receiving ROM as first TPO-RA and attaining sustained responses (P=0.073), while switching TPO-RA negatively predicted TFR (P=0.010). In univariate analysis with logistic regression, switching TPO-RA was associated with a 6.4 risk of not achieving TFR (P= 0.019). The overall comparison of the Kaplan-Meier curves indicated an association (log-rank P=0.041) among the initial TPO-RA that was prescribed and the probability of TFR (Fig 1A), but the estimated median time to achieve TFR was not reached for either TPO-RA. When switching and initial TPO-RA were considered, patients receiving ROM and not experiencing switching were the best performing group in terms of achieving TFR; the median time to taper off the drug and sustain platelet responses was 3.3 years (95% CI 2.7-4.0 years) (Fig. 1B). Conclusion. In this observational research analysis, we have tried to minimize the possible bias of some studies that could mistakenly attribute TPO-RA induced TFR, when in fact it may be the natural course of the underlying disease. By analyzing a group of chronic ITP patients with a particularly poor baseline prognosis (median time from diagnosis 5.5 years) we address potential confounding variables by disease severity. Our data show that assuring that long duration under TPO-RA therapy is provided (median of 3.3 years), one half of chronic ITP patients treated with ROM and not undergoing switching can achieve TFR. Disclosures Mingot-Castellano: Roche: Consultancy; Bayer: Consultancy; Novartis: Consultancy; Takeda: Consultancy; Sobi: Consultancy; Amgen: Consultancy; Novonordisk: Consultancy; CSL Behring: Consultancy. Jarque:Abbie: Consultancy, Speakers Bureau; Alexion: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Grifols: Consultancy; Gilead: Consultancy, Speakers Bureau; CellTrion: Consultancy; Celgene: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Servier: Speakers Bureau; Roche: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; MSD: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Shire: Consultancy, Speakers Bureau; Shionogi: Consultancy, Speakers Bureau. Campos:Novartis: Speakers Bureau; Amgen: Speakers Bureau. Lopez Fernandez:Amgen: Consultancy, Speakers Bureau. Casado:Amgen: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Álvarez Roman:Amgen: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Speakers Bureau; Sobi: Consultancy, Speakers Bureau; Takeda: Research Funding; NovoNordisk: Consultancy, Speakers Bureau.