ALBERT

Description: Key Points Increased immune suppressors and PD-1 abrogates effector responses in CML patients at diagnosis. Enhanced net effector immune responses and decreased PD-1 and immune suppressors may promote sustained deep molecular response in CML.

Description: Abstract 3383 There are three currently identified secondary resistance mechanisms observed in chronic myeloid leukemia (CML) patients receiving tyrosine kinase inhibitors (TKIs). These include overexpression of drug-efflux proteins (ABCB1 and ABCG2), increased BCR-ABL expression, and mutations in the kinase domain (KD) of BCR-ABL. We investigated the interplay between these three modes of resistance in vitro, as well as looking for other mechanisms. Three CML blast crisis cell lines (K562, its ABCB1-overexpressing variant, K562 Dox, and KU812) were cultured in gradually increasing concentrations of imatinib (IM) to 2μ M, or dasatinib (DAS) to 200nM. Two IM-resistant K562 lines were established, both with increased IC50s for IM (from 13.7μ M in naïve cells, to ~50μ M), as well as increased IC50s for DAS and nilotinib (NIL). No cell-surface expression of ABCB1 or ABCG2 was observed, nor were KD mutations present. However, BCR-ABL expression was seen to steadily increase in both lines from 178% in naïve cells, to ~380% and 1200% in the resistant lines, suggesting this was the major mode of resistance. However, when a DAS-resistant K562 culture was generated the T315I mutation emerged. Studies of the intermediate stages of resistance revealed that BCR-ABL overexpression occurred in a step-wise fashion, peaking at 1915% in the 3.5nM intermediate, but then dropping significantly to ~1000% in the 5nM intermediate (P=0.0003). BCR-ABL expression then stabilised at this level, and the T315I mutation was subsequently detected. Thus, it appears that BCR-ABL overexpression was the first mechanism of resistance detectable, but was followed by the emergence of a KD mutation which had a clear selective advantage. This sequential selection was observed a further four times: in a DAS-resistant K562 Dox culture, and in three IM-resistant KU812 cultures. BCR-ABL expression in the DAS-resistant K562 Dox culture increased from 186% in naïve cells to 540% in the final culture. Studies of intermediate cultures revealed that BCR-ABL expression peaked at 850% in the 55nM intermediate, but then dropped significantly to ~500% in the 75nM intermediate (P=0.004). This drop in BCR-ABL expression coincided with the appearance of the V299L mutation. Interestingly, the K562 Dox DAS-resistant line also displayed resistance to NIL and IM, likely conferred by BCR-ABL overexpression as the 55nM intermediate (with the highest BCR-ABL expression levels) had the highest IC50s for NIL and IM, while the 75nM intermediate (with the V299L mutation) had increased IC50DAS but lower NIL and IM IC50s. Thus, BCR-ABL overexpression was the primary event, followed by the KD mutation. Likewise in three IM-resistant KU812 cultures, BCR-ABL expression levels rose from 443% in naïve cells, to peak levels of 6210%, 10,448% and 990% respectively, followed by drops in expression which coincided with the appearance of compound KD mutations, and the F359C mutation respectively (Table). In contrast, three IM-resistant K562 Dox cells were not found to have any KD mutations, nor BCR-ABL overexpression. Instead, the primary cause of resistance in these lines appears to be a further increase in ABCB1 expression. All three lines had increased IC50s for IM (from 12μ M in naïve cells, to ~27μ M), NIL (from 400nM to ~1000nM) and DAS (from 100nM to 〉625nM). The addition of PSC833 (an ABCB1 inhibitor) decreased the IM, NIL, and DAS IC50s for all three resistant lines to the level of the naïve control (~3μ M, ~250nM and ~10nM respectively), indicating that ABCB1 expression, facilitating active efflux of the drugs, is the primary mechanism of resistance in these lines. We have demonstrated that KD emergence is a stochastic event, as the same mutation did not always occur twice, however BCR-ABL and ABCB1 overexpression were more likely to arise recurrently in predisposed lines. Notably, different TKIs elicited different resistant mechanisms, but all were BCR-ABL dependent. Furthermore, all resistant lines showed cross-resistance to the three TKIs tested (IM, DAS and NIL), suggesting that currently available TKIs share the same susceptibilities to drug resistance. Table 1. Summary of resistance mechanisms detected in three cell lines exposed to IM or DAS. ✓ = yes; × = no. Culture condition K562 K562 Dox KU812 IM IM DAS IM IM IM DAS IM IM IM Resistance to 3 TKIs ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ KD Mutation x x T315I x x x V299L E450QE459KE470K E459KE462KE466E F359C Increased BCR-ABL ✓ ✓ ✓ x x x ✓ ✓ ✓ ✓ Increased ABCB1 x x x ✓ ✓ ✓ x x x x Disclosures: White: Novartis Pharmaceuticals: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Hughes:Novartis Pharmaceuticals: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Research Funding, Speakers Bureau.

Description: We hypothesized that immune responses contribute to deep BCR-ABL molecular responses in chronic phase chronic myeloid leukaemia (CML) patients on tyrosine kinase inhibitors (TKI). We studied 32 CML patients; 16 at diagnosis, patients treated with imatinib (n=20), nilotinib (n=9) or dasatinib (n=3). Methodology: The effector immune responses of Natural Killer (NK) cells were characterized by flowcytometry and functional analysis by CD107a degranulation assay. Cytotoxic T lymphocyte (CTL) responses to leukaemia-associated antigens (LAAs) WT1, BMI-1, PR3 and PRAME were quantified by interferon-gamma ELISPOT using peptide libraries of 15-mer peptides overlapping by 11 amino acids spanning the entire protein, or HLA-A0201 specific peptides in HLA-A0201+ patients. Immune suppressor regulatory T cells (Treg), Myeloid Derived Suppressor Cells (MDSC), Programmed cell death-1 (PD-1) expression on T cells, NK cells, B cells and monocytes, and major B cell subsets were extensively characterized by flowcytometry. Results: Patients in deep molecular response (MR4.5; BCR-ABL

Description: Introduction A correlation exists between innate immune responses and outcomes in cancer treatment, and immunological features may be prognostic biomarkers of TKI response in chronic phase CML patients (CML-CP pts). Marin et al (Leuk 2012) found KIR2DS1 to be associated with CCyR, OS and PFS, while Kreutzman et al (Exp Hem 2012) showed KIR2DL5A/B to be associated with MMR. We examined the prognostic significance of KIR (Killer Immunoglobulin-like Receptor) genotypes in CML-CP pts in the TIDEL-II study who received upfront treatment with imatinib and early switching to nilotinib for suboptimal responses. Method TIDEL-II is a multicentre, single arm prospective ALLG trial for de novo CP-CML pts in 2 equal sequential cohorts of 105 pts in each. All pts started on imatinib (IM) 600mg OD. Pts were monitored for achievement of time dependent molecular targets (BCR-ABL 10%, 1% and 0.1% IS at 3, 6 and 12 months respectively). Pts in cohort I (C1) who failed these targets were dose escalated to IM800. Pts failing to achieve these targets subsequently, or who were already on IM800, switched to nilotinib 400mg BID (NIL). Pts in cohort II (C2) who failed their time dependent targets switched to NIL regardless of their IM dose. Switching to NIL was also permitted for Grade III/IV or persistent Grade II non-hematological toxicity or loss of response. Baseline samples were available for 148 pts, on which KIR genotyping was done retrospectively using the KIR Genotyping SSP Kit (Invitrogen, Carlsbad, CA). Molecular response and survival outcomes were analysed as stratified by early molecular response (EMR, BCR-ABL ≤10% at 3 months), gender, Sokal Index, age and KIR genotype. Results The 24 month MMR rate was 73% and EMR failure was 12%. Overall and progression-free survival (PFS; events = transformation to AP/BC + any death) was 94% and 93% at 4 years respectively. Failure free survival (FFS; events in PFS in addition to loss of MMR / CCyR and failure according to 2013 ELN criteria) was 76% at 4 years. In a competing risk univariate analysis, EMR correlated with MMR achievement as expected, but not Sokal, age or sex. This analysis also showed inferior MMR achievement for pts with KIR2DL5B (HR 0.423, p=0.00041), KIR2DL2 (HR 0.607, p=0.0048) and KIR2DS3 (HR 0.547, p=0.0027) genotypes. The number of pts with these alleles were 31 (21%), 83 (56%) and 44 (30%) respectively. As predicted from the population distribution of KIR genotypes, these 3 alleles were in strong linkage disequilibrium. Only KIR2DL5B (2DL5B) and EMR remained independent predictors of MMR in a multivariate model (2DL5B positive HR 0.52, p=0.034). KIR2DL5B positivity was also independently associated with inferior rates of MR4.5 (HR 0.42, p=0.031) (Fig. top panels). Four year PFS was inferior for pts positive for 2DL5B (86% vs 97%, p=0.04), as was FFS (67% vs 80%, p=0.05) (Fig. lower panels). There was no correlation between 2DL5B status and EMR achievement. However pts negative for 2DL5B who had EMR failure were more likely to achieve MMR, 7/13 pts (54%), compared with 0/5 2DL5B positive pts (all 18 pts switched to NIL). This was not statistically significant (p=0.09), due to the small numbers. In patients who achieved EMR, 94% of 100 2DL5B negative pts achieved MMR, vs 76% of the 25 2DL5B positive pts. Among pts with EMR failure, 2DL5B positivity was associated with a trend for inferior survival at 4 years. PFS was 91% vs 98% and FFS was 80 vs 84% for 2DL5B pos vs neg pts respectively. Conclusion KIR genotypes had previously been correlated with achievement of CCyR, PFS and OS. Here, we have demonstrated that the KIR2DL5B allele correlated with lower rates of MMR and MR4.5 in a multivariate analysis, even in a treatment schema allowing patients failing early molecular targets to be treated with nilotinib, independent of EMR. Additionally, KIR2DL5B positivity was associated with inferior PFS and FFS. Multiple studies have shown the prognostic significance of EMR, as has our data. KIR genotyping information may further refine the prognosis of patients failing to achieve EMR. Prognostic markers available at CML-CP diagnosis, such as KIR genotypes, may have clinical utility. Furthermore, the KIR genotype may provide useful information in combination with other biomarkers and could be incorporated into future prognostic scoring systems for CML-CP. Figure 1 Figure 1. Disclosures Yeung: 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. White:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Ariad: Research Funding. Branford:Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding. 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; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Description: Key Points KIR2DL5B is associated with poor molecular response and transformation-free survival in CML patients enrolled to the TIDEL-II study. KIR genotyping would select out high risk CML patients at baseline and allow better targeting of novel interventions.