ALBERT

Description: Abstract 1690 Background: We have previously identified that low OCT-1 activity (OA) is a poor prognostic indicator in CP-CML patients treated with imatinib (IM). Importantly, a very low OA (OA≤4ng/200,000 cells) is associated with a significant risk of poor molecular response, kinase domain mutations and transformation. The TIDEL II strategy of early intervention, via dose escalation and/or switch to nilotinib (NIL) may reduce the incidence of poor response/therapeutic failure in CP-CML patients, particularly those with very low OA. Methods: Patients in Cohort I (n=105) of the TIDEL II trial were switched to NIL for either IM intolerance, or failure to demonstrate a clinical benefit from IM dose escalation which was triggered by failure to achieve time dependent molecular targets: ≤10% BCR-ABL by 3 m, ≤1% BCR-ABL by 6 m or ≤0.1% BCR-ABL by 12 m. In Cohort II (n=105) patients failing to achieve these molecular targets were switched directly to NIL without prior IM dose intensification. All patients, where possible, had OA measured at diagnosis. Only therapeutic changes prior to 24 months, and patients with a minimum of 6 months exposure to NIL are considered in this analysis. Results: (Table 1) Cohort I: Median follow-up 30 months. The overall rate of major molecular response (MMR) by 12 months was 66%. There was a significant difference in the rate of MMR between patients with low OA (n=49) compared to those with higher OA (n= 54): 49% vs 76%, p=0.007. The overall rate of MMR by 24 months was 81%. Again, patients with low OA (n=46) achieved MMR at a significantly lower rate compared to those with higher OA (n=54): 65% vs 91%, p= 0.003. Thirty patients have switched to NIL, 19/30 because of IM intolerance (av. time on IM 8.5m.) and 11/30 because of molecular target failure (av. time on IM 12.8m). 14/14 intolerant patients not in MMR at the time of switch have achieved MMR on NIL with an average log reduction of 2.8, and 9/18 have achieved CMR. In contrast, 1/9 patients switched for molecular target failure has achieved MMR on NIL, with an average log reduction of 0.65. Importantly, 3/19 withdrew from study due to CML related events. Cohort II: Median follow-up 12 months. To date, 22/105 patients have switched to NIL and have a minimum of 6 months follow-up: 11 for intolerance and 11 for molecular target failure. All patients switched for intolerance achieved and/or maintained MMR on nilotinib, with an average log reduction of 2.89. In contrast, 1/11 patients switched for molecular target failure achieved MMR, with an average log reduction of 0.95 and CCyR has been achieved in 5/10 patients not previously in CCyR. 2/11 of these patients have withdrawn from study. There was a significant difference in the time of switch to NIL, and the length of imatinib exposure between the 2 cohorts for intolerance, and a significant difference between the cohorts in the length of IM exposure for patients with target failure. However, this did not translate to a significant difference in molecular response between the 2 cohorts, suggesting the length of prior IM exposure is not a determinant of subsequent NIL response. Importantly in both cohorts, the OA of those patients switched to NIL based on molecular target failure was significantly lower than that of those who switched for intolerance (p=0.007 and p=0.003) and those patients remaining on IM (p=0.004). Conclusion: Switch to NIL significantly improves response in IM intolerant patients. The majority of patients who switch for molecular target failure on IM do not subsequently achieve MMR on NIL. This suggests that a low OA may delineate a group of CP-CML patients intrinsically insensitive to TKI therapy, for whom switch to NIL either following IM dose intensification (Cohort 1) or as a primary strategy (Cohort II) may not result in an improvement in response. A different first-line strategy may be more effective for this poor risk subgroup. Disclosures: White: Novartis Pharmaceuticals: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Slader:Novartis Pharmaceuticals: Employment, Equity Ownership. Yeung:Novartis Pharmaceuticals: Research Funding; BMS Oncology: Research Funding. Osborn:Novartis Pharmaceuticals: Research Funding; BMS Oncology: Research Funding. Mills:Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Sponsorship to professional meetings; BMS Oncology:. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ARIAD: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Interpatient variability in intracellular uptake and retention (IUR) of imatinib may be due to variable function of the OCT-1 influx pump. OCT-1 activity was measured in pretherapy blood from chronic myeloid leukemia (CML) patients by calculating the difference in IUR of [14C]-imatinib with and without OCT-1 inhibition. Of patients with higher than median (high) OCT-1 activity, 85% achieved major molecular response (MMR) by 24 months, versus 45% with no more than a median (low) OCT-1 activity. Assessing patients receiving 600 mg imatinib per day and those averaging fewer than 600 mg over 12 months of therapy revealed patients with high OCT-1 activity achieved excellent molecular response regardless of dose, whereas response of patients with low OCT-1 activity was highly dose dependent. Of patients with low OCT-1 activity who received fewer than 600 mg, 45% failed to achieve a 2-log reduction by 12 months, and 82% failed to achieve a MMR by 18 months, compared with 8% and 17% in the cohort with high OCT-1 activity and dose less than 600 mg/day (P = .017 and P = .022). OCT-1 activity is an important determinant of molecular response to imatinib, with predictive value closely linked to dose. This pretherapy assay identifies patients at greatest risk of suboptimal response where dose intensity is critical, and those likely to respond equally well to standard dose imatinib.

Description: We have previously demonstrated significant interpatient variability in the IC50imatinib, a measure of the intrinsic sensitivity of a patient to imatinib induced kinase inhibition. Furthermore, this measure is predictive of the achievement of major molecular response (MMR 〉 3 log reduction in BCR-ABL) in de-novo CML patients treated with imatinib (n=60)1. In an expanded patient pool (n=116) we now perform an evaluation of the IC50 as a predictor of response, and address the IC50imatinib as a guide to dose selection. Samples were obtained with informed consent from de novo CML patients enrolled to either the TIDEL (600mg imatinib) or TOPS (randomised 400mg vs 800mg imatinib) trials. Blood was collected pre therapy, and the IC50 was performed as previously1. Outcome data was assessed using Kaplan Meier Analysis and the log rank test was used to assess statistical significance. In our previous analysis the IC50imatinib was divided about the median value for the cohort (0.6μM) into low and high IC50, with a significantly greater proportion of patients with low IC50imatinib achieving MMR by 12 months. In this expanded patient pool, we confirm this finding (0.95uM achieve MMR on 400mg, and that this is statistically significantly when compared to all other groups. At 600mg while there is no overall significant difference there is a statistically relevant difference between groups 1, 2 and 4 as indicated. In contrast, at 800 mg the effect of IC50imatinib is overcome. MMR by 12 months Total 400mg 600mg 800mg p value Group1 0.50.70.95μM 32% (28) 0% (7)* 22% (9)* 58% (12) 0.016 P value 0.042 0.018 0.108 0.778 Table 1: Dividing the patients into quartile based on the IC50 imatinib and assessing the Impact of dose on the achievement of MMR by 12 month. *p value 0.7μM fail to achieve a 2 log reduction when treated with 400mg (IC50 0.7μM 33% p=0.034), and 600mg (IC50 0.7μM 22% p=0.036). However, there is no significant difference in the 800mg patient cohort (IC50 0.7μM 14% p=0.79). This analysis confirms that the IC50imatinib, is predictive of imatinib response. Patients with an IC50imatinib 0.7μM) may benefit from higher dosing regimens (p=0.012). Thus, the accurate assessment of IC50imatinib could support dose optimization strategy for patients with a suboptimal response.

Description: The ability to predict which CML patients respond poorly to imatinib may facilitate a more aggressive approach to therapy in these patients. We have used western blotting to assess the phosphotyrosine content of the adaptor protein Crkl (p-Crkl) -a major substrate for BCR-ABL. PB was collected from 57 newly diagnosed CML patients prior to the initiation of therapy and dose dependent inhibition of Crkl phosphorylation, in the presence of imatinib, was measured following incubation of primary cells for 2 hours with varying doses of imatinib. Using this assay we were able to determine that the median IC50 for imatinib was 0.6 uM (R 0.375–1.8uM). For statistical analysis IC50’s were then grouped into low and high relative to the median, and compared to molecular response achieved at both 3 and 12 months. Using Log Rank Survival Analysis there was a significant difference in the probability of achieving a 2 log depletion of BCR-ABL (measured by quantitative real-time Q-PCR) by 3 months between the two groups (Low IC50 30% n=31, High IC50 4% n=26; p=0.023 n=57). In addition the probability of achieving a 3 log reduction of BCR-ABL at 12 months (MMR, major molecular response) was significantly different (Low IC50 47%, High IC50 23%, p=0.034 ). We therefore assessed whether the IC50 was a surrogate measure of the Sokal Score or was an independent predictor of response. When we examined the median Sokal scores of both the low and high IC50 groups we found that there was no significant difference between the two groups (Low IC50 = 1.0, High IC50 = 0.9 p=〉0.05 ) suggesting that IC50 was not a surrogate measure of Sokal. Log Rank survival analysis revealed that while Sokal Score did not predict for a 2 log depletion at 3 months (Low (43% n=32) vs Intermediate (21%n=25) vs High (21% n=29) p〉0.05 ) it was predictive of a greater probability of MMR at 12 months (Low 53% vs Intermediate 42% vs High 21% p=0.044 ). The combined effect of these parameters was assessed: Probability of achieving a Major Molecular Response. All Low Sokal Intermediate Sokal High Sokal Note: Not all Sokal Scores available at the time of analysis. Low IC50 47% n=30 67% n=9 50% n=6 17% n=12 High IC50 23% n=23 20% n=10 22% n=9 0% n=4 p-value 0.034 0.037 〉0.05 〉0.05 Evidence presented here suggests that the IC50 level measured in blood cells from CML patients prior to imatinib therapy provides a statistically significant indicator of response at both early and late timepoints. Further to this, the combined measures of Sokal Score and IC50 provide a powerful tool for prediction of molecular response, which clearly delineates those patients groups who have a low probability of achieving a major molecular response. Preliminary investigations into the components of both Sokal and IC50 which may be contributing to these findings have highlighted a group of patients (n=7) who have a high blast percentage (5 to 21%) and low IC50. Unlike the majority of patients with low IC50 these patients all fail to achieve MMR. When these patients are removed from the low IC50 group the probability of MMR in the remaining patients is 71%. Further investigations into underlying mechansims for the inter-patient variation in IC50 are currently being undertaken.

Description: Abstract 2189 Poster Board II-166 Introduction. The organic cation transporter 1 (OCT-1) is the major active influx transporter for imatinib in PB mononuclear cells (MNC) from CML patients. We have previously demonstrated that the functional activity of the OCT-1 protein (OCT-1 Activity, OA) in MNC from chronic phase (CP) CML patients is highly variable (mean: 7.2 ng/200,000 cells, standard deviation: 6.4). We also demonstrated that the MNC OA is significantly related to a patient's sensitivity to imatinib-induced kinase inhibition, and their subsequent molecular response. In addition, we have recently demonstrated that OA is predictive of event free and transformation free survival to 5 years (White ASH submitted 2009). This is of a greater importance for those patients on lower doses of imatinib (below 600mg). For these patients who have a low MNC OA, 82% failed to achieve a major molecular response (MMR) by 18 months of imatinib treatment, as opposed to 17% for patients with a high MNC OA (p=0.022). Recently, we have shown that the OA in CML CD34+ cells is significantly lower than that found in mature CML cells (mean CD34+: 3.9, CD34-: 11.6 ng/200,000 cells, p

Description: The human organic cation transporter-1 (hOCT-1) is the primary active influx protein for imatinib in BCR-ABL1+ cells. A low functional OCT-1 activity (OA) in peripheral blood mononuclear cells (MNCs) at diagnosis is associated with poorer subsequent molecular response and inferior outcomes in chronic phase chronic myeloid leukaemia (CP-CML) patients. We have previously demonstrated that diclofenac, an antagonist of peroxisome proliferator-activated receptor-γ (PPARγ), can significantly increase OA in BCR-ABL1+ cells. In the current study, we systematically assess the correlation between PPARγ and OA using leukemic cell lines and primary MNCs from untreated de novo CP-CML patients. Given the critical role of PPARγ in cell differentiation and the varying levels of PPARγ expression in different cell types, the present study also explored the relationship between the OA and myeloid-specific markers (CD14, CD15 and CD16) in CP-CML patient MNCs. Our results demonstrate that OA in BCR-ABL1 positive and negative leukemic cell lines (KU812, HL60 and HL60-BCRABL1) were significantly decreased with PPARγ agonists (GW1929, rosiglitazone) and increased with antagonists (GW9662, T0070907) (Table 1). Similarly, OA in PPARγ-transduced K562 cells (16.03 ng/200,000 cells) was significantly reduced when compared with empty vector control (22.73 ng/200,000 cells, p=0.028). In primary MNCs isolated from untreated CP-CML patients with high OA, a consistent inhibitory effect on OA was observed following the treatment with PPARγ agonists in all 5 cases. The average OA was significantly reduced by GW1929 (from 7.88 to 5.21 ng/200,000 cells, p=0.0075) or rosiglitazone (to 4.65 ng/200,000 cells, p

Description: Abstract 3250 Poster Board III-1 Preclinical studies of imatinib set the paradigm of continuous Bcr-Abl kinase inhibition for optimal response in chronic myeloid leukemia (CML). However, the clinical success of once daily dasatinib, despite its short serum half life, implies that intermittent inhibition of Bcr-Abl kinase activity is sufficient for clinical response. In vitro studies also demonstrated that short-term intense (≥90%) Bcr-Abl kinase inhibition triggers cell death in BCR-ABL + cell lines, demonstrating their oncogene addiction. However, the effect of short-term intense kinase inhibition on CD34+ CML progenitors is not studied. Clinical, mathematical modelling and in vitro studies suggest that leukemic stem cells (LSC) are difficult to eradicate and hence the majority of CML patients may not be cured with tyrosine kinase inhibitors (TKI). Inadequate Bcr-Abl kinase inhibition has been postulated to cause refractoriness of LSC to TKI's. This may be due to increased expression of ABCB1 and ABCG2 efflux proteins, or the quiescent state of LSC. However, the phenomenon could be independent of Bcr-Abl kinase activity. In vivo leukemic progenitors live in a cytokine rich environment which may be providing a mechanism for Bcr-Abl independent resistance. We have assessed the impact of short-term intense Bcr-Abl kinase inhibition on CML cell lines and CML CD34+ primary cells in the presence and absence of cytokines. In CML cell lines, short-term (cells were cultured with dasatinib for 30 min and following thorough drug washout, cells were recultured in drug free media for 72 hr) intense Bcr-Abl kinase inhibition with 100 nM dasatinib triggers cell death. In CML-CD34+ cells 30 min of culture with 100 nM dasatinib (n=13) or 30 μM IM (n=7) reduced the level of p-Crkl (surrogate marker of Bcr-Abl kinase activity) by 97±3% and 96±4% respectively. In the presence of either a six growth factors cocktail (6-GF; n=10) or GM-CSF (n=11) or G-CSF (n=4) alone, despite 97% inhibition of p-Crkl, short-term culture with 100 nM dasatinib (D100ST) reduced colony forming cells (CFC) by only 24%, 32% or 5%, respectively. However without cytokines, D100ST reduced CML-CD34+ CFCs by 70%. Consistent with the results observed with dasatinib, short-term culture with 30 μM imatinib (IM) (n=3) also reduced 90% CFC in the absence of cytokines but by only 38% in the presence of 6-GF. These results suggest that in CML-CD34+ cells, GM-CSF, G-CSF or 6-GF mediate Bcr-Abl independent TKI resistance. It is possible that cytokines may be promoting cell survival via signalling pathways that are refractory to dasatinib. To examine this possibility, we assessed the effect of D100ST on p-STAT5 signalling in CML-CD34+ cells, in the presence and absence of GM-CSF, G-CSF or 6-GF. STAT5 was constitutively phosphorylated in CML-CD34+ cells, and in the absence of cytokines, D100ST reduced the p-STAT 5. STAT5 phosphorylation was not inhibited by D100ST when cells were cultured with 6-GFs or GM-CSF however, the combination of D100ST and a Janus kinase (Jak) inhibitor dramatically reduced p-STAT5. Similarly, in the presence of GM-CSF (32.35±5.16% vs. 68.33±14.90%) or G-CSF (58.13±13 vs. 94.68±21.12) combination of D100ST and JAK inhibitor significantly reduced CFC compared to D100ST only. Thus our data suggest that in contrast to CML cell lines, primary CML progenitors may not be completely dependent on the BCR-ABL oncogene and that activation of the cytokine mediated JAK-2/STAT-5 pathway may circumvent the need for BCR-ABL signalling for maintenance of survival. Thus a therapeutic strategy based on short-term intense kinase inhibition may have limited success unless critical redundant cytokine-induced survival pathways are also inhibited. We postulate that blockade of cytokine signalling along with short-term intense Bcr-Abl kinase inhibition with a potent second generation TKI may provide a novel strategy to eradicate primitive CML cells. Fig 1 In CML-CD34+ cells, Jak kinase inhibition abrogates the rescuing effect of cytokines on cell death induced by BCR-ABL blockade: In the absence of cytokines (No GF, n=11) short-term culture with 100 nM dasatinib (D100ST) reduced CFCs by 67% of control, however in the presence of 6-GFs (n=10), GM-CSF (n=10) or G-CSF (n=4) it could reduce CFCs by only 24%, 32% or 5% of control respectively (B) In the presence of GM-CSF (n= 4) or G-CSF (n= 4), combination of Jak inhibition and D100ST reduced CFC compared to dasatinib alone. Fig 1. In CML-CD34+ cells, Jak kinase inhibition abrogates the rescuing effect of cytokines on cell death induced by BCR-ABL blockade: In the absence of cytokines (No GF, n=11) short-term culture with 100 nM dasatinib (D100ST) reduced CFCs by 67% of control, however in the presence of 6-GFs (n=10), GM-CSF (n=10) or G-CSF (n=4) it could reduce CFCs by only 24%, 32% or 5% of control respectively (B) In the presence of GM-CSF (n= 4) or G-CSF (n= 4), combination of Jak inhibition and D100ST reduced CFC compared to dasatinib alone. Disclosures: White: Novartis and Britol-Myers Squibb: Research Funding. Hughes:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Tyrosine Kinase Inhibitors (TKIs) result in excellent responses in Chronic Myeloid Leukaemia (CML) patients. However, secondary resistance is observed in ~35% of patients, mostly due to Bcr-Abl kinase domain (KD) mutations. Overexpression of the efflux transporter ABCB1 is a known mediator of primary resistance to imatinib (IM) and nilotinib (NIL). ABL001, a potent allosteric inhibitor, binds to the myristate pocket of the Bcr-Abl KD. In this study, we modelled ABL001 resistance in vitro with particular focus on ABCB1 and ABCG2. ABL001 sensitivity was evaluated in BCR -ABL1 + cell lines K562, K562-Dox (ABCB1 overexpressing) and K562-ABCG2 (transfected with ABCG2 overexpression vector). Also, K562, K562-Dox and KU812 cells were made resistant to ABL001 by culturing long term in increasing concentrations of ABL001 (increased once 〉80% survival in culture for 〉10 days). Parental ABL001 naive cells were maintained in parallel culture. Initial onset of resistance was characterised by significantly increased IC50ABL001 based on AnnexinV/7-AAD cytotoxicity assays performed at each ABL001 dose escalation. Resistance mechanisms were interrogated during escalation and once 10 μM was reached (based on clinically achievable doses). Protein expression levels of efflux transporters ABCB1/ABCG2 were examined by flow cytometry; KD mutation sequencing and Bcr-Abl protein quantitation were performed. K562-Dox and K562-ABCG2 cells demonstrated significantly increased IC50ABL001 compared with parental K562 control cells: 256 and 299 nM respectively vs 23 nM, p

Description: Background Mutation of genes linked to hematologic cancer have recently been reported in CML and are associated with early progression and resistance (Reviewed in Branford, Kim Leuk 2019). The mutations comprise single nucleotide variants (SNVs) and small insertions/deletions (indels), plus gene fusions and large focal gene deletions. In 39 patients (pts) in blast crisis (BC), all had at least 1 cancer gene mutation, including fusions in 33%: partner genes MLL, RUNX1, IKZF1, MECOM and CBFB. 50% of the fusions were novel and some were present at chronic phase diagnosis. BCR-ABL1 mutations rarely occurred as the sole mutant. NGS offers critical information for resistance assessment. For many clinical purposes, targeted DNA sequencing (seq) using panels of specific disease related genes is the most cost effective screening choice. However, this strategy could miss relevant fusions and deletions. Aim To determine whether an RNA based approach is more informative than DNA for detecting a broad range of mutations. Method A hybridization capture NGS gene panel was developed to target 126 genes relevant for myeloid/lymphoid leukemia. In a pilot study, DNA and RNA derived from 5 leukemia cell lines with well characterized mutations, including fusions and deletions, were panel sequenced. An additional 6 cell lines were sequenced using RNA, plus 49 pt samples with RNA stored for up to 14.6 years: 45 at diagnosis and 4 at BC/resistance. Six of these pt samples had prior whole exome and/or whole transcriptome seq. We used total RNA that detected intronic splice region variants from pre-spliced RNA. SNVs/indels were called from DNA/RNA with FreeBayes. Manta called focal deletions from DNA. Known and novel RNA fusions and novel splice junctions were detected using the STAR aligner. Gene expression used edgeR. Results For the 5 cell lines with DNA versus (v) RNA seq, SNVs/indels were reliably called in RNA, with a strong positive correlation of mutant allele frequency: DNA v RNA, r = 0.93. Two TP53 small deletions of 26 and 46 bp were not called in RNA, but were instead detected as novel RNA splice junctions. Read counts were 5.2 fold higher for RNA than DNA at sites of clinically relevant mutants, consistent with enrichment of seq read depth proportional to expression. Overall, RNA revealed a higher number of relevant mutants than DNA: RNA = 49 v DNA = 37, Fig A-C. Notably, the functional effect of splice region disrupting mutants and large focal deletions were evident by novel RNA splicing, Fig D-F. In the total 11 cell lines tested with RNA, all 13 reported fusions were called, including BCR-ABL1 and RUNX1, MLL, ETV6 and CBFB fusions. For 7 cell lines with variants described in the COSMIC Cell Lines Project, 23/23 cancer gene SNVs/indels were called, plus 7 cancer gene SNVs/indels not reported. These were verified by DNA seq. 15 gene deletions were evident by atypical RNA splicing and verified by DNA seq: IKZF1, CDKN2A/B, PAX5, BTG1, RB1 and NCOR1. Five other cell lines had verified CDKN2A deletions that were evident by loss of gene expression, Fig G. Two BTG1 deletions were not detected. For the 6 pt samples re-sequenced by the RNA panel, 8/8 verified fusion transcripts were detected with a 31 fold enrichment of read counts. 11/11 cancer gene SNVs/indels were called and 3/4 gene deletions. The exception was a CDKN2A deletion not detected by novel splicing but evident as loss of expression, Fig G. Seven other cancer gene SNVs were found at low allele frequency, including a resistant BCR-ABL1 mutation at 1.7% in the oldest sample. Of the 43 diagnosis samples without prior NGS, BCR-ABL1 transcripts were detected in all. BCR-ABL1 genomic breakpoints were called at base pair resolution in 39, 91%. Two pts had mutated ASXL1 at diagnosis and both failed imatinib by 9 months with mutant BCR-ABL1. By gene expression analysis, all but 1 of the total 45 diagnosis samples clustered together. The exception was a pt who transformed to lymphoid BC at 6 months that clustered with the lymphoid cell lines and lymphoid BC pts, Fig H. Conclusion RNA gene panel seq demonstrated enhanced sensitivity and an increased yield of clinically relevant mutations compared with DNA panel seq. A single RNA assay has the capacity to detect SNV/indels, known and novel gene fusions, focal deletions and the likely functional effect of splice disrupting mutations. RNA panel seq is a valuable tool for the comprehensive assessment of mutations that drive CML treatment failure and drug resistance. Disclosures Branford: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Qiagen: Consultancy, Honoraria; Cepheid: Consultancy, Honoraria. Shanmuganathan:Gilead: Other: Travel Support; Janssen: Other: Travel Support; Amgen: Other: Travel Support; Bristol-Myers Squibb: Honoraria, Other: Travel Support; Novartis: Honoraria, Other: Travel Support. Scott:Celgene: Honoraria. Hughes:Novartis, Bristol-Myers Squibb: Consultancy, Other: Travel; Novartis, Bristol-Myers Squibb, Celgene: Research Funding.

Description: Background Mutated cancer genes in patients (pts) with TKI failure and blast crisis (BC) CML have recently been described. RUNX1 mutations, namely single nucleotide variants (SNVs) and indels, were the most frequently detected besides BCR-ABL1 [reviewed in Branford, Kim Leuk 2019]. They were found in ~18% of pts, although splice variants were rarely described. RNA splicing events were associated with focal deletion of IKZF1 and RUNX1 in TKI resistant pts that were identified by copy number analysis and RNAseq [Branford Blood 2018]. Novel splicing associated with mutation of cancer genes is an unexplored area of study in resistance. RNA sequencing can assess the functional effect of splice site variants, which lead to splicing errors due to the use of alternative or cryptic splice sites and cause alterations to protein function. Aim We determined whether novel splicing can identify cancer genes with potential altered function. Methods RNAseq analysis was performed for 48 pts at diagnosis and 33 at BC using a protocol that preserved intron-retaining precursor RNA. Coverage of intron-exon borders was sufficient to detect intronic splice region variants. The STAR aligner was used to bioinformatically collate unannotated RNA splice junctions. 54 cancer genes were assessed and aberrant splice events were filtered based on the number of samples in which a splice junction occurred. Manual inspection of the splice junctions was performed using the Integrative Genomics Viewer. This approach identified previously verified aberrant splicing associated with IKZF1 and RUNX1 deletions. Results Ten previously undetected novel splice junctions were revealed in 9/33 pts (27%) in BC within key tumor suppressor genes CDKN2A/B (5), RB1 (1), ATM (1), and RUNX1 (3). The aberrant splicing pattern of CDKN2A and RB1 (Fig A/B) in 6 pts suggested large deletions, as previously described in our cohort with IKZF1 and RUNX1 deletions. Breakpoints associated with deletions ranging from 53 to 181 Kb were detected in the 5 pts with CDKN2A aberrant splicing. Similarly, a 90 Kb deletion of exons 18-27 of the RB1 gene led to the aberrant splicing. The pts transformed to lymphoid BC (median 5 months). 4 of these 6 pts were tested at diagnosis and the deletions were not detected, indicating they were gained at resistance. The aberrant splicing patterns of ATM and RUNX1 did not predict large deletions. These were related to somatic SNVs at canonical splice sites in ATM and in 2 of the pts with RUNX1 aberrant splicing. A splice acceptor site SNV in ATM resulted in skipping of exon 61 (Fig C) and protein truncation. This novel SNV has not been reported in any population or somatic variant database. Two pts in myeloid BC at 28 and 48 months after diagnosis had an identical somatic RUNX1 mutation at the canonical splice donor site of exon 5. The SNV was not detectable prior to imatinib treatment in both pts. The splice site SNV led to activation of a cryptic splice site within exon 5 in both pts (Fig D), which predicted premature termination. While this mutation is novel, an adjacent intronic SNV occurs in familial platelet disorder, leading to activation of the same cryptic splice site. The atypical RUNX1 splicing of the 3rd patient was associated with retention of 55 bp of intron 6 as a cryptic exon (Fig E), leading to protein truncation. A deep intronic SNV identified at lymphoid BC at 6 months of imatinib was detected near the cryptic exon by RNAseq and verified as somatic by DNA Sanger sequencing. This was predicted to activate cryptic RNA splicing elements and lead to intron sequence retention in a RUNX1 transcript. We sequenced the diagnosis sample using an RNA-based gene panel method under development that provides enhanced sensitivity of variant detection. The same pattern of atypical splicing was observed and the intronic SNV was present at low level. The RUNX1 mutation at diagnosis may have contributed to early BC. To our knowledge this is the first report of a RUNX1 truncating variant in CML involving a cryptic exon. Conclusion Enhanced bioinformatic analysis of RNAseq data has revealed a high proportion of pts with truncating mutations in cancer genes indicated by novel RNA splicing (27% pts in BC). Using RNA-based sequencing allows an evaluation of the potential functional effect of variants that are not apparent by DNA-based mutation analysis. We suggest that future studies include RNA sequencing to detect a broader spectrum of mutations associated with TKI resistance. Disclosures Shanmuganathan: Gilead: Other: Travel Support; Janssen: Other: Travel Support; Amgen: Other: Travel Support; Bristol-Myers Squibb: Honoraria, Other: Travel Support; Novartis: Honoraria, Other: Travel Support. Yeung:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria. Scott:Celgene: Honoraria. Hughes:Novartis, Bristol-Myers Squibb, Celgene: Research Funding; Novartis, Bristol-Myers Squibb: Consultancy, Other: Travel. Branford:Cepheid: Consultancy, Honoraria; Qiagen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau.