Abstract: Patients up to age 70 years with CML treated within a decentralized health care setting had a relative survival close to 1.0. Sokal, but not EUTOS, score at diagnosis predicted overall and relative survival in a population-based cohort of patients with CML.

Abstract: In the present study we combined interferon (IFN) and hydroxyurea (HU) treatment, intensive chemotherapy and autologous stem cell transplantation (SCT) in newly diagnosed chronic myelogenous leukemia patients aged below 56 years, not eligible for allogeneic SCT. Patients who had an HLA-identical sibling donor and no contraindication went for an allogeneic SCT (related donor, RD). After diagnosis, patients not allotransplanted received HU and IFN to keep WBC and platelet counts low. After 6 months patients with Ph-positive cells still present in the bone marrow received 1–3 courses of intensive chemotherapy. Those who became Ph-negative after IFN + HU or after 1–3 chemotherapy courses underwent autologous SCT. Some patients with poor cytogenetic response were allotransplanted with an unrelated donor (URD). IFN + HU reduced the percentage of Ph-positive metaphases in 56% of patients, and 1 patient became Ph-negative. After one or two intensive cytotherapies 86 and 88% had a Ph reduction, and 34 and 40% became Ph-negative, respectively. In patients receiving a third intensive chemotherapy 92% achieved a Ph reduction and 8% became Ph-negative. The median survival after auto-SCT (n = 46) was 7.5 years. The chance of remaining Ph-negative for up to 10 years after autologous SCT was around 20%. The overall survival for allo-SCT RD (n = 91) and URD (n = 28) was almost the same, i.e. ≈60% at 10 years. The median survival for all 251 patients registered was 8 years (historical controls 3.5 years). The role of the treatment schedule presented in the imatinib era is discussed.

Abstract: Biologic and clinical observations suggest that combining imatinib with IFN-α may improve treatment outcome in chronic myeloid leukemia (CML). We randomized newly diagnosed chronic-phase CML patients with a low or intermediate Sokal risk score and in imatinib-induced complete hematologic remission either to receive a combination of pegylated IFN-α2b (Peg–IFN-α2b) 50 μg weekly and imatinib 400 mg daily (n = 56) or to receive imatinib 400 mg daily monotherapy (n = 56). The primary endpoint was the major molecular response (MMR) rate at 12 months after randomization. In both arms, 4 patients (7%) discontinued imatinib treatment (1 because of blastic transformation in imatinib arm). In addition, in the combination arm, 34 patients (61%) discontinued Peg–IFN-α2b, most because of toxicity. The MMR rate at 12 months was significantly higher in the imatinib plus Peg–IFN-α2b arm (82%) compared with the imatinib monotherapy arm (54%; intention-to-treat, P = .002). The MMR rate increased with the duration of Peg–IFN-α2b treatment ( 〈 12-week MMR rate 67%, 〉 12-week MMR rate 91%). Thus, the addition of even relatively short periods of Peg–IFN-α2b to imatinib markedly increased the MMR rate at 12 months of therapy. Lower doses of Peg–IFN-α2b may enhance tolerability while retaining efficacy and could be considered in future protocols with curative intent.

Abstract: The inhibition of oncogenic BCR-ABL1 kinase with tyrosine kinase inhibitors (TKIs) has significantly improved the prognosis of CML. Recent reports suggest that approximately 40 % of CML patients who have achieved optimal therapy response (complete molecular remission, CMR) can stop imatinib treatment without recurrence of detectable BCR-ABL1 transcripts. However, no predictive prognostic factors for successful therapy discontinuation have yet been identified. We therefore set up an immunological substudy in the ongoing pan-European EURO-SKI stopping study. We aimed to identify predictive biomarkers for relapse and non-relapse after TKI discontinuation. In addition, we aimed to understand more on the mechanisms of immune surveillance in CML and to study the effects of TKI treatment on the immune system. Materials and methods Patients in deep molecular remission (MR4, BCR-ABL 〈 0,01% IS) for at least one year and with TKI treatment for at least 3 years were eligible for the clinical study. Basic lymphocyte immunophenotyping (the proportions and absolute numbers of NK-, T- and B-cells) was performed at the university hospital laboratories at the time of therapy discontinuation, and 1, 6, and 12 months after the TKI discontinuation. In a proportion of patients a more detailed immunophenotypic (analysis of CD45RA, CD57, CD27 and CD62L expressions) and functional analyses were done from fresh blood samples in a central immunology laboratory (Helsinki) at the same time points. The cytotoxicity of NK-cells was studied by measuring the direct killing of target cells (K562) and by the degranulation assay (CD107a/b expression). The secretion of Th1 type of cytokines IFN-γ/TNF-α was studied from both T- and NK-cells. Results Thus far the basic lymphocyte subclass measurement has been analyzed from 62 patients who have discontinued TKI treatment within the EURO-SKI study. Functional analyses have been performed from 30 patients. 60 patients have used imatinib before treatment discontinuation and 2 patients dasatinib. At baseline, before the treatment discontinuation both CD4+ and CD8+ T-cell counts were within the normal range (median CD4+ 0.73x 109/L, range 0.11-2.4x 109/L; CD8+ 0.35x 109/L, 0.07-1.92 x 109/L). The TKI stop had no significant numerical or functional effect on T-cells, and at 1 month time-point the median T-cell counts were unchanged (CD4+ 0.73x 109/L; CD8+ 0.35x 109/L). Similarly, at the baseline, the median NK-cell count was within a normal range (0.26 x 109/L, range 0.04-1.04 x 109/L), and no significant change was observed 1 month after stopping the treatment (median 0.29 x 109/L). Furthermore, at the baseline and at the 1-month time-point the cytotoxicity of NK-cells and the cytokine secretion of T- and NK-cells did not significantly differ from the healthy controls when all patients were considered as a one group. However, when patients were divided in two groups based on the relapse status, the patients who eventually relapsed had significantly fewer NK-cells already at the baseline (Figure A; absolute count 0.18x 109/L vs. 0.32 109/L, p=0.008; proportions 11% vs. 21%, p=0.001). The phenotype of NK-cells also differed between the two groups, and the patients who relapsed had less NK-cells expressing CD57 (median 58% vs. 69%, p=0.046) and CD16 (median 67% vs. 83%, p=0.018) on the cell surface. Furthermore, the cytotoxicity of NK-cells was impaired in patients who failed to discontinue the TKI treatment successfully and no killing activity was observed in their samples (Figure B; alive K652 cells after co-incubation with effector cells 100% vs. 88%, p=0.07). No clear differences were observed in the function or the numbers of T-cells between relapsing and non-relapsing patients. Conclusions The NK-cell numbers and their function may predict disease relapse after TKI discontinuation. This may have impact on the future stopping trials. In addition, it further illustrates the importance of the immune system in the successful long-term treatment of CML. Disclosures: Ekblom: Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Hjorth-Hansen:Pfizer, BMS: Honoraria, Travel expenses Other. Porkka:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Richter:Bristol-Myers Squibb: Consultancy, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau. Mustjoki:Novartis: Honoraria; BMS: Honoraria, Research Funding.

Abstract: Background: Recent reports suggest that approximately 40% of CML patients who have achieved sustained complete molecular remission are able to stop TKI treatment without disease relapse. However, there are no predictive markers for successful therapy discontinuation. Therefore, we set up an immunological sub-study in the ongoing pan-European EURO-SKI stopping study. Our aim was to identify predictive biomarkers for relapse/non-relapse and to understand more on the mechanisms of immune surveillance in CML. Methods: The EURO-SKI study started in 2012, and patients included were at least three years on TKI and at least one year in MR4 or deeper before the study entry. Basic lymphocyte immunophenotyping (the number of NK-, T- and B-cells) was performed at the time of therapy discontinuation and 1, 6, and 12 months after the TKI stop and in case of relapse (defined as loss of MMR, BCR-ABL1 〉 0.1% IS). In addition, from a proportion of patients more detailed immunophenotypic and functional analyses (cytotoxicity of NK-cells and secretion of Th1 type of cytokines IFN-γ/TNF-α) were done at the same times. Results: Thus far 119 Nordic patients (imatinib n=105, dasatinib n=12, nilotinib n=2) who have discontinued TKI treatment within the EURO-SKI study have been included in the lymphocyte subclass analysis (results are presented from patients who have reached 6 months follow-up). Immunophenotyping analysis demonstrates that imatinib treated patients who were able to maintain remission for 6 months (n=36) had increased NK-cell counts (0.26 vs. 0.15x109cells/L, p=0.01, NK-cell proportion 18.9% vs. 11%, p=0.005) at the time of drug discontinuation compared to patients who relapsed early (before 5 months n=22). Furthermore, the phenotype of NK-cells was more cytotoxic (more CD57+ and CD16+cells and less CD62L+cells), and also their IFN-γ/TNF-α secretion was enhanced (19.2% vs. 13%, p=0.02). Surprisingly, patients who relapsed more slowly (after 5 months, n=16) had similar baseline NK-cell counts (0.37x109cells/L), NK-cell proportion (21.2%), and phenotype and function as patients, who were able to stay in remission. No differences in the NK-cell counts were observed between patients who had detectable or undetectable BCR-ABL1 transcripts at the baseline (0.22 x109cells/L vs. 0.31 x109cells/L, p=0.61). Interestingly, NK-cell count was higher in patients with low Sokal risk score than in patients with intermediate risk (0.33 x109cells/L vs. 0.20 x109cells/L, p=0.04). Furthermore, there was a trend that male patients had a higher proportion of NK-cells than females (21.6% vs. 15.7%, p=0.06). Pretreatment with IFN-α or the duration of imatinib treatment did not have an effect on NK-cell count or proportion. In comparison to the imatinib group, dasatinib treated patients had higher NK-cell counts at the baseline (median 0.52x109cells/L vs. 0.26x109cells/L, p=0.02), and also the proportion of CD27 (median 50% vs. 16%, p=0.01) and CD57 expressing (median 79% vs. 74%, p=0.05) NK-cells was higher. The follow-up time of dasatinib treated patients is not yet long enough to correlate the NK-cell counts with the success of the treatment discontinuation. The absolute number of T-cells or their function did not differ significantly between relapsing and non-relapsing patients at the time of treatment discontinuation. However, both CD4+ and CD8+ T-cells tended to be more mature in patients who stayed in remission compared to patients who relapsed early (CD4+CD57+CD62L- median 5.7% vs. 2.4%, p=0.06, CD8+CD62L+CD45RA+ 13% vs. 26.7%, p=0.05). The analysis of follow-up samples showed that in patients who stayed in remission the Th1 type cytokine (IFN-γ/TNF-α) secretion of CD8+T-cells increased at 6 months compared to baseline (23.6 vs. 18.5%, p=0.07). Same phenomenon was observed in the late relapsing group at relapse compared to baseline (37.9 vs. 13.5%, p=0.03). No similar increase was observed in the early relapsing group. Conclusions: Low NK-cell numbers and poor cytokine secretion may predict early disease relapse after TKI discontinuation. However, patients who relapse later have high numbers of normally functioning NK-cells. Further research (detailed phenotypic analysis of NK- and T-cells including activating and inhibitory receptors and immune checkpoint molecules) and correlation of biomarker data with clinical parameters are ongoing to understand the ultimate determining factors of relapse. Disclosures Själander: Novartis: Honoraria. Hjorth-Hansen:Novartis: Honoraria; Bristol-myers Squibb: Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Porkka:BMS: Honoraria; BMS: Research Funding; Novartis: Honoraria; Novartis: Research Funding; Pfizer: Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Abstract: Background: Tyrosine kinase inhibitors (TKIs) have significantly improved the treatment of CML. Even though TKI treatment is generally not considered curative, recent studies have shown that nearly half of CML patients who have achieve good and durable responses are able to stop the TKI treatment. However, patients who have successfully discontinued TKI treatment still have residual disease. We hypothesized that the immune system plays a role in treatment free remission (TFR), and our preliminary results in the EURO-SKI trial showed that patients who relapse early after imatinib discontinuation have decreased numbers and frequencies of NK cells. In EURO-SKI trial relapse was defined as the loss of major molecular response (MMR). We now aimed to analyze in more detail the phenotype and function of the NK cells in order to understand their role in TFR. Methods: Lymphocyte subclass analysis (the number of NK-, T- and B-cells) was performed at the time of therapy discontinuation and 1 month after the imatinib discontinuation in patients participating in the EURO-SKI stopping trial in the Nordic countries (n=105, results are presented from patients who have reached 6 months follow-up). More detailed immune phenotype and functional assays (NK-cell degranulation and secretion of Th1 type of cytokines IFN-γ/TNF-α) were analyzed from a proportion of patients (n=31). Results: Imatinib treated patients remaining in remission for 6 months (non-relapsing, n=48, median age 60,5 years) displayed an increased amount of NK cells at the time of drug discontinuation (18.6% vs. 11.0%, p=0.02, NK-cell count 0.25 x109 cells/L vs. 0.184 x109 cells/L m, p=0.059) compared to patients who relapsed early (before 5 months, n=29, median age 60,5 years). Furthermore, the NK cell frequency in non-relapsing patients was even higher than in healthy controls (11.5%, n=48, p=0.001). T and B cell counts and frequencies showed no differences between the groups. Detailed analysis of the NK cell compartment displayed a more mature phenotype for the NK cells in non-relapsing patients. Larger frequencies of NK cells from early relapsing patients was CD56bright compared to non-relapsing patients (4.8% vs. 2.7% of CD56 NK cells, p=0.04). Furthermore, patients who had higher frequencies of CD56bright NK cells than median had decreased TFR at 6 months (42%) compared to patients with lower frequency (70%, p=0.01). In addition, there was a trend towards more CD57pos (78% (n=21) vs. 66% (n=10), p=0.09) CD56dim NK cells in non-relapsing patients. To further study the mature NK cells in non-relapsing patients, recently identified markers (FceRgneg, PLZFneg, SYKneg, EAT-2neg) for adaptive NK cells were analyzed. Interestingly, there was a trend that non-relapsing patients had higher frequencies of adaptive-like NK cells. For example, non-relapsing patients had more CD56dim NK cells that had down regulated EAT-2 (2.8% (n=6) vs. 1.3% (n=5) of lymphocytes, p=0.03) and more CD56dim NK cells expressing NKG2D (11.2% vs. 2.6% of lymphocytes, p=0.02) and NKp46 (13.6% vs. 3.9% of lymphocytes, p=0.05). Moreover, after imatinib discontinuation the expression of transcription factor Eomes increased in the CD56dim NK cells of the early relapsing group (baseline MFI 2045 vs. 1 month 3480, p=0.06), while in non-relapsing group it seemed to even decrease (baseline MFI 2273 vs. 1 month 1980, p=0.13) pointing towards an adaptive phenotype. No significant differences between the groups were observed when degranulation against K562 cell line was studied. However, CD16neg NK cells from non-relapsing patients responded to K562 stimulation by secreting more TNFα/IFNγ compared to the early relapsing patients (21% vs. 13% of CD56pos CD16neg NK cells, p=0.01). Furthermore, patients whose CD16neg NK cells had higher than median TNFα/IFNγ secretion when stimulated with K562 cells showed an increased TFR at 6 months (78%) compared to patients who had lower TNFα/IFNγ secretion than median (37%, p=0.005). Conclusions: CML patients who successfully discontinued imatinib therapy displayed a higher number and frequency of peripheral blood mature, adaptive-like NK cells capable of secreting cytokines TNFα/IFNγ relative to relapsing patients. How such NK cells may contribute to maintenance of treatment free remission is still unknown. Nonetheless, our results warrant further clinical studies with NK-cell modulating agents. Disclosures Muller: Novartis: Honoraria, Other: Consulting or Advisory Role, Research Funding; ARIAD Pharmaceuticals Inc.: Honoraria, Other: Consulting & Advisory Role, Research Funding; BMS: Honoraria, Other: Consulting or Advisory Role, Research Funding. Hjorth-Hansen:Novartis: Honoraria; Ariad: Honoraria; Bristol-Myers Squibb: Research Funding; Pfizer: Honoraria, Research Funding. Saussele:Pfizer: Honoraria, Other: Travel grant; BMS: Honoraria, Other: Travel grant, Research Funding; Novartis Pharma: Honoraria, Other: Travel grant, Research Funding; ARIAD: Honoraria. Mahon:ARIAD: Consultancy; Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy. Porkka:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Richter:Ariad: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria. Mustjoki:the Finnish Cancer Societies: Research Funding; Pfizer: Honoraria, Research Funding; Academy of Finland: Research Funding; Sigrid Juselius Foundation: Research Funding; Finnish Cancer Institute: Research Funding; Signe and Ane Gyllenberg Foundation: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.