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: Introduction: The introduction of continuous tyrosine kinase inhibitor (TKI) treatment has dramatically improved progression-free survival for chronic phase chronic myeloid leukaemia (CML) patients. This success, however, has put the issue of long-term drug toxicity and safety into focus. Recent data from clinical studies have indicated an increased risk of cardiovascular events (CVE), including peripheral arterial occlusive disease, in CML patients receiving treatment with the TKIs nilotinib or ponatinib, as compared to imatinib (Giles et al, Leukemia 2013; Kim et al, Leukemia 2013; Cortes et al, New England Journal of Medicine 2013; FDA communication 2013). This study used data retrieved from Swedish population-based registries to estimate the frequency of CVE in CML patients, particularly those treated with imatinib and the 2nd generation TKIs nilotinib and dasatinib. Methods: We identified all incident cases between 2002 and 2012 in the Nationwide Swedish CML register. All patients who were in blast crisis or accelerated phase at time of diagnosis were excluded. All patients were followed untill death, emigration or 31st December 2012. For all CML patients a comparison cohort was established, matched to be of the same age and sex as the CML cohort, with 5 control subjects per CML patient. By means of record linkage with the nationwide Swedish patient register both cohorts were followed for the occurrence of adverse cardiovascular outcomes. Two sets of relative risks (expressed as incidence rate ratios; IRRs) of cardiovascular and venous thromboembolic disease were computed. In a first step CML patients were compared to the control population. In a second step, restricted to CML patients ever treated with TKIs, CML patients on different TKI treatments were compared. Patients could be treated with several TKIs during their follow-up, and events would only be attributable to the TKI used during the time period. Both analyses were adjusted for age, sex and calendar period. The second analysis was also adjusted for Sokal risk score. Results: A total of 896 CML patients were included and followed during a median of 4.2 years (Table I). The main outcome data are presented in Table II. A total of 23 venous thrombotic events (VTE) and 60 arterial thrombotic events were detected in the CML patient cohort during follow-up. Compared with the general population, this corresponded to significantly increased risks. In particular, deep venous thrombosis and “other arterial thromboses” were more common among CML patients (IRR 2.41 95% CI 1.29-4.52 and IRR 3.50 95% CI 1.36-9.04, respectively). Assessing risks associated with particular TKIs, we noted that treatment with any of the 2nd generation TKIs nilotinib or dasatinib, as compared to imatinib, was associated with a significantly increased occurrence of myocardial infarction (IRR 2.98 95% CI 1.05-8.49 and IRR 2.89 95% CI 1.20-7.00, respectively). Notably, there were no differences in the occurrence of CVE between the different patient groups before CML diagnosis. Conclusion: These data, derived from a large population-based Swedish cohort, provide evidence of an increased risk of both venous and arterial thrombotic events among CML patients and that patients on 2nd generation TKIs, as compared to imatinib, may be at increased risk of myocardial infarction. Further analyses will assess whether these differences may reflect patient selection and characterstics, rather than drug-related factors. Meanwhile, risk factors for CVE should be observed and considered in the TKI treatment of CML. Figure 1 Figure 1. Figure 2 Figure 2. * Footnote: the number of events may not add up because of occurrence of more than one type of vascular event in one subject. The number of events in the analysis within the CML cohort is lower than in the comparison with the general population because of exclusion of patients who were never treated with TKIs in the former analysis. Disclosures Björkholm: Novartis: Research Funding; Shire: Research Funding; Merck: Research Funding; Amgen: Honoraria, Research Funding; Pfizer: Research Funding; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Akinon: Honoraria; Nordic Nanovector: Honoraria. Själander:Novartis: Honoraria. Richter:Ariad: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria.

Abstract: Background: The etiology of chronic myeloid leukemia (CML) is essentially unknown with high doses of ionizing radiation being the only well established risk factor. We have recently published two large population-based studies showing an increased prevalence of other malignancies prior, as well as subsequent to a diagnosis of CML (Gunnarsson et al, Br J Haematol. 2015 Jun; 169(5): 683-8 and Gunnarsson et al, Leukemia. 2016 Jul; 30(7): 1562-7). One may therefore speculate that CML patients may have an increased congenital or acquired susceptibility to develop cancer. In the former case, one would expect an increased prevalence of malignancies among first-degree relatives (FDR) to CML patients. In a previous report based on the Swedish Cancer Registry, no increased aggregation of malignancies was detected among family members to CML patients diagnosed between 1958 and 2004 (Bjorkholm et al, Blood. 2013 Jul 18; 122(3): 460-1). However, a more strict definition of CML (requiring e.g. thepresence of a Philadelphia chromosome or the BCR/ABL fusion gene) was introduced with the updated WHO classification in 2002, making subsequently diagnosed CML cohorts more homogenous. Materials and methods: Aiming to establish the prevalence of malignancies among FDR of a large and well-defined contemporary CML cohort in Sweden compared to carefully matched controls, we have used four large Swedish population based registers. To identify Swedish patients with CML diagnosed between 2002 and 2013, we used the Swedish CML Register to which all CML patients diagnosed January 1st 2002 and later are reported. FDR were identified by use of the Swedish Multi-Generation Register, which comprises information about parent-sibling-offspring relationships of persons that has been registered in Sweden at some time since 1961 and born later than 1932. By linking this cohort to the Swedish Cancer Register, we retrieved information about malignancies for each FDR. Each CML patient was matched with five, age-, gender- and county of residence-matched controls, selected from the Swedish Total Population Register. All controls had to be alive and free of CML at the time of CML diagnosis for the matching CML patient. To calculate odds ratio (OR) and 95% confidence intervals (CI), conditional logistic regression were used. Results: In the Swedish CML register, 984 patients were identified. Among them 184 patients were excluded due to a birth year prior to 1932. Among the 800 remaining CML patients, 4 287 FDR were identified and included in the analysis (parents: 1 346, siblings: 1 497 and children: 1 444). Correspondingly, 20 930 matched controls were included in the analysis. In total, 611 malignancies were identified among the FDR of CML patients compared to 2844 in the control group yielding an OR of 1.057 (95% CI 0.962 - 1.162). Neither hematological malignancies nor solid tumors were increased in the CML-FDR group (Table 1). Notably, none of the FDRs in the CML-FDR group had a CML diagnosis. Conclusions: Using data from four large Swedish population based registers and based on the fate of more than 4 000 FDR of 800 CML patients diagnosed in the modern era of cytogenetics, as well as closely matched CML-free controls, we show that there is no familial aggregation of malignancies in FDRs of patients with CML. These results suggest that a hereditary predisposition to develop cancer is unlikely to be a part of the pathogenesis of CML. Disclosures Höglund: Akinion Pharmaceuticals: Consultancy; Janssen-Cilag: Honoraria. Lambe:AstraZeneca: Other: Stock Ownership ; Pfizer: Other: Stock Ownership . Richter:Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding. Själander:ARIAD: Consultancy.

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.

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: Background: Since continuous treatment with tyrosine kinase inhibitors (TKIs) has dramatically improved the survival of patients with chronic myeloid leukemia (CML), it is of interest to examine the possible risk of long-term adverse events. Previous studies have presented conflicting results regarding risk of second malignancies. Our aim was to examine the development of second malignancies (except acute myeloid or lymphoblastic leukemia, myelodysplastic syndromes or non-melanoma skin cancer) in CML chronic phase patients diagnosed after the introduction of TKI treatment. Materials and methods: We studied the development of second malignancies in 868 patients diagnosed with CML in chronic phase 2002 to 2011 using the Swedish CML register, cross-linked to the Swedish Cancer register. Each patient was followed from the time of CML diagnosis until death from any cause, date of allogeneic hematopoietic stem cell transplantation (SCT) or end of study on December 31, 2011, whichever came first. SCT was used as an endpoint because of the well established increased risk of second malignancies after this procedure. Standardized Incidence Ratios (SIR) were calculated to assess the risk of a second malignancy by dividing the number of observed second malignancies with the number of expected malignancies in the Swedish population, using data from the Swedish Cancer Register. The expected numbers of malignancies were determined by dividing the CML population according to 5-year age groups, sex, region of residence (6 regions) and calendar year. The number of person-years in each stratum was multiplied with the incidence of malignancies or deaths found in the corresponding strata in the general population. Results: With a median follow-up of 3.7 (range 0-9.9) years, 65 (7.5%) patients developed 75 second cancers (non-hematologic), 49 of these of invasive type. Compared to expected rates in the background population matched by age, sex, region of residence (6 regions) and calendar year, the risk of second malignancies was significantly higher in the CML cohort, with a Standardized Incidence Ratio (SIR) of 1.5 (95 % CI 1.13-1.99). SIR before and after the second year following diagnosis of CML was 1.6 (95 % CI 1.004-2.38) and 1.5 (95 % CI 0.98-2.11), respectively. Looking at CML subpopulations, the increased risk of developing a second malignancy reached statistical significance for females (SIR: 1.8; 95 % CI 1.18-1.99), but not for males (SIR: 1.3; 95 % CI 0.85-1.91), and for patients above 60 years of age at diagnosis (SIR: 1.5; 95 % CI 1.05–1.96). Assessment of risk by cancer type was hampered by small numbers. However, the data at hand indicate an increased risk for gastrointestinal cancer (SIR: 3.0; 95 % CI 1.60-5.16), as well as nose and throat cancer (SIR: 37.1; 95 % CI 7.46-108.40), table 1. Conclusions: Utilizing large, population-based registries with data accumulated during the TKI era, our results indicate that CML patients, compared to the normal control population, are at an 50% increased risk of developing a second malignancy. Similar SIR before and after the second year following the diagnosis of CML may indicate that these findings are linked to the CML disease itself, rather than to the TKI treatment. Further studies and longer follow-up seem however warranted. Physicians caring for CML patients should be aware of signs and symptoms of other malignancies in this patient population. Table 1 Standardized Incidence Ratios for second malignancies (excluding cases of non-melanoma skin cancer, AML, ALL and MDS) among 868 Swedish CML patients diagnosed between 2002 and 2011. Total follow up time 3293 person-years (median 3.7 years). Variable Observed Expected SIR (Observed/Expected) 95 % CI for SIR Overall 52 34 1.5 1.13–1.99 Men 26 20 1.3 0.85–1.91 Women 26 14 1.8 1.18–2.66 Age 〈 60 years 10 5 1.9 0.89–3.42 Age ³ 60 years 42 28 1.5 1.05–1.96 Second cancer type Prostate 14 8 1.8 0.96–2.94 Gastrointestinal 13 4 3.0 1.60–5.16 Gynecological 4 1 3.6 0.98–9.30 Nose and Throat 3 0,1 37.1 7.46-108.40 Lung 2 2,7 0.7 0.08-2.67 Breast 4 4,2 0.98 0.26-2.45 Disclosures Björkholm: Novartis: Research Funding; Shire: Research Funding; Merck: Research Funding; Amgen: Honoraria, Research Funding; Pfizer: Research Funding; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Akinon: Honoraria; Nordic Nanovector: Honoraria. Richter:Ariad: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria. Själander:Novartis: Honoraria.

Abstract: Clinical trials show that tyrosine kinase inhibitor (TKI) treatment can be discontinued in selected patients with chronic myeloid leukaemia (CML). Although updated CML guidelines support such procedure in clinical routine, data on TKI stopping outside clinical trials are limited. In this retrospective study utilising the Swedish CML registry, we examined TKI discontinuation in a population‐based setting. Out of 584 patients diagnosed with chronic‐phase CML (CML‐CP) in 2007–2012, 548 had evaluable information on TKI discontinuation. With a median follow‐up of nine years from diagnosis, 128 (23%) discontinued TKI therapy (≥1 month) due to achieving a DMR (deep molecular response) and 107 (20%) due to other causes (adverse events, allogeneic stem cell transplant, pregnancy, etc). Among those stopping in DMR, 49% re‐initiated TKI treatment (median time to restart 4·8 months). In all, 38 patients stopped TKI within a clinical study and 90 outside a study. After 24 months 41·1% of patients discontinuing outside a study had re‐initiated TKI treatment. TKI treatment duration pre‐stop was longer and proportion treated with second‐generation TKI slightly higher outside studies, conceivably affecting the clinical outcome. In summary we show that TKI discontinuation in CML in clinical practice is common and feasible and may be just as successful as when performed within a clinical trial.

Abstract: Given that tyrosine kinase inhibitors ( TKI s) have dramatically improved the survival of patients with chronic myeloid leukaemia ( CML ), we were interested in examining the possible risk of long‐term adverse events, such as the emergence of other neoplasms. Therefore, we studied the development of second malignancies in 868 patients diagnosed with CML between 2002 and 2011 using the Swedish CML register, cross‐linked to the Swedish Cancer register. With a median follow‐up of 3·7 (range 0–9·9) years, 65 (7·5%) patients developed 75 second malignancies (non‐haematological), 52 of which were of the invasive type. Compared to expected rates in the background population, the risk of second malignancies was higher in the CML cohort, with a standardized incidence ratio ( SIR ) of 1·52 (95% CI 1·13–1·99). The SIR before and after the second year following diagnosis of CML was 1·58 and 1·47, respectively. Among specific cancer types, gastrointestinal and nose and throat cancer were significantly increased. Founded on a population‐based material, our results indicate that CML patients treated in the TKI era are at an increased risk of developing a second malignancy, with indications that this risk may more likely be linked to CML itself rather than to the TKI treatment.