Supplementary Figure 3. Effects of kinase inhibitor treatment on expression of the pro-apoptotic molecule BIM.

Supplementary Figure 2. Additional K562 BCR-ABL Western Blot densitometry.

Supplementary Figure 1. Representative flow cytometry plots and gates.

Supplementary Tables 1-3. Supplementary Table 1: Changes in manual differential white cell counts over time under treatment with BRAF/MEK inhibitor combination therapy for melanoma. Supplementary Table 2. BCR-ABL monitoring during treatment. Supplementary Table 3. Leukocyte sub-populations as defined by flow cytometry.

Early relapse of hematological malignancies after allogeneic hematopoietic stem cell transplantation (alloSCT) is associated with a poor prognosis. Biomarkers predictive of relapse may allow pre-emptive therapies to prevent relapse. Donor T-cell chimerism is associated with relapse after myeloablative alloSCT, however its sensitivity and specificity have not been described. We investigated day 100 donor T-cell chimerism (D100chim) alone or in combination with disease risk index (DRI) as a predictor of relapse free survival (RFS) at 6months after myeloablative alloSCT. Methods: We retrospectively analyzed outcomes of patients who underwent myeloablative alloSCT for any hematological malignancy at our institution from 2000-2016 with minimum 6 months follow-up. D100chim was measured by PCR analysis of short tandem repeats on peripheral blood T-cells. DRI was assigned as previously reported (2014). The primary outcome was RFS at 6 months post-alloSCT, treated as a binary end point. D100chim was stratified into categories separated by 10 percentage points from 55-95%. P < .05 was interpreted as statistically significant. Results: 207 patients were analyzed with median follow-up 61 months (Table 1). Twenty (9.7%) patients relapsed or died within 6 months of alloSCT (18 relapse, 2 died before relapse). Mean D100chim was 91.5% (±SEM .96%). There was no difference in D100chim between different conditioning regimens (P = .09). A D100chim threshold of 65% was most significantly associated with 6month RFS (P = .0097) (Figure 1). DRI (OR 3.50, P = .01) and BuCy conditioning (OR 6.3, P = .02) were also significantly associated with 6month RFS, while T-cell depletion, stem cell source and donor type did not demonstrate associations. In a multivariable model, D100chim ≤ 65% and DRI were independently associated with 6month RFS.Table 1Patient DemographicsMedian Age(Range)42(17-60)Sex(M/F)124/83Hematological malignancy(%)AML90(43.5)MDS13(6.3)MPN22(10.6)ALL52(25.1)NHL17(8.2)CLL9(4.3)Other4(1.9)DRILow/Intermediate173(83.6)High/Very High34(16.4)DonorSibling112(54.1)Matched unrelated donor95(45.9)T-cell depletion85(41.1)ConditioningCyTBI65(31.4)TBI-VP1646(22.2)BuCy96(46.4) Open table in a new tab We analyzed the performance of D100chim at a threshold of 65% as a predictive biomarker of early RFS. The specificity of D100chim for 6month RFS was 95.7% however sensitivity was poor at 20%. We combined DRI and D100chim in an algorithm with high/very high DRI and D100chim ≤ 65% each scoring 1 point. The specificity of a total score of 1 or greater remained high at 82% with an improved sensitivity of 55%. Conclusion: D100chim is highly specific but lacks sensitivity for RFS at 6months after alloSCT. An algorithm incorporating DRI and D100chim improves the sensitivity for RFS at 6months and is a more clinically useful tool than either predictor alone.

Donor T cell chimerism is associated with relapse outcomes after allogeneic stem cell transplantation (alloSCT) for acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). However, measures of statistical association do not adequately assess the performance of a prognostic biomarker, which is best characterized by its sensitivity and specificity for the chosen outcome. We analyzed donor T cell chimerism results at day 100 (D100chim) after myeloablative alloSCT for AML or MDS in 103 patients and determined its sensitivity and specificity for relapse-free survival at 6 months (RFS6) and 12 months (RFS12) post-alloSCT. The area under the receiver operating characteristic curve for RFS6 was .68, demonstrating only modest utility as a predictive biomarker, although this was greater than RFS12 at .62. Using a D100chim threshold of 65%, the specificity for RFS6 was 96.6%; however, sensitivity was poor at 26.7%. This equated to a negative predictive value of 88.5% and positive predictive value of 57.1%. Changing the threshold for D100chim to 75% or 85% modestly improved the sensitivity of D100chim for RFS6; however, this was at the expense of specificity. D100chim is specific but lacks sensitivity as a prognostic biomarker of early RFS after myeloablative alloSCT for AML or MDS. Caution is required when using D100chim to guide treatment decisions including immunologic manipulation, which may expose patients to unwarranted graft-versus-host disease.

Abstract Purpose: Targeted MEK inhibition is an emerging therapy in a number of solid tumors. It holds particular promise in BRAF V600E mutation–positive malignant melanoma, where constitutive activation and cell growth through the MAP kinase (MAPK) pathway is well established. In vitro and preclinical research indicates that MAPK pathway activation is important in chronic myeloid leukemia (CML) leukemogenesis; however, the potential of MEK inhibition has not yet been investigated clinically in the setting of such hematologic malignancies. Experimental Design: We report a case of complete hematologic response of CML to MEK inhibition in a patient with synchronous metastatic melanoma, who received treatment with combination BRAF and MEK1/2 inhibitors. We studied the effects of these agents on proliferation and outgrowth of myeloid precursors, and longitudinal shifts in peripheral blood phenotyping during the course of treatment. A model cell line system was used to examine the effects of dabrafenib and trametinib on MAPK and BCR–ABL1 signaling. Results: After 35 weeks on treatment with BRAF and MEK inhibitors, complete hematologic response was observed without recourse to BCR–ABL1–targeted therapy. MEK inhibition was principally responsible for impaired proliferation of both mature and primitive myeloid precursors, as well as growth and hemoglobinization of erythroid precursors. Paradoxical activation of the MAPK pathway was seen in response to BRAF inhibitor therapy but this was easily overcome by clinically relevant doses of concurrent MEK inhibitor. Conclusions: These studies suggest that further evaluation of the optimal MAPK targeting approach is warranted to extend therapeutic options in CML. Clin Cancer Res; 21(23); 5222–34. ©2015 AACR.

Purpose: Targeted MEK inhibition is an emerging therapy in a number of solid tumors. It holds particular promise in BRAF V600E mutation-positive malignant melanoma, where constitutive activation and cell growth through the MAP kinase (MAPK) pathway is well established. In vitro and pre-clinical research indicates that MAPK pathway activation is important in chronic myeloid leukemia (CML) leukemogenesis however the potential of MEK inhibition has not yet been investigated clinically in the setting of such hematological malignancies. Experimental design: We report a case of complete hematological response of CML to MEK inhibition in a patient with synchronous metastatic melanoma, who received treatment with combination BRAF and MEK1/2 inhibitors. We studied the effects of Research. on April 12, 2017. © 2015 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 22, 2015; DOI: 10.1158/1078-0432.CCR-15-0393 ANDREWS et al. CLINICAL RESPONSE OF CML TO MEK INHIBITION 4 4 these agents on proliferation and outgrowth of myeloid precursors, and longitudinal shifts in peripheral blood phenotyping during the course of treatment. A model cell line system was used to examine the effects of dabrafenib and trametinib on MAPK and BCR-ABL1-signalling. Results: After 35 weeks on treatment with BRAF and MEK inhibitors, complete hematologic response was observed without recourse to BCR-ABL1-targeted therapy. MEK inhibition was principally responsible for impaired proliferation of both mature and primitive myeloid precursors, as well as growth and hemoglobinization of erythroid precursors. Paradoxical activation of the MAPK pathway was seen in response to BRAF inhibitor therapy but this was easily overcome by clinicallyrelevant doses of concurrent MEK inhibitor. Conclusions: These studies suggest that further evaluation of the optimal MAPK targeting approach is warranted to extend therapeutic options in CML. Research. on April 12, 2017. © 2015 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 22, 2015; DOI: 10.1158/1078-0432.CCR-15-0393 ANDREWS et al. CLINICAL RESPONSE OF CML TO MEK INHIBITION 5 5 INTRODUCTION Chronic myeloid leukemia (CML) is almost universally characterized by a reciprocal translocation between the breakpoint cluster region (BCR) gene on chromosome 22, and the Abelson murine leukemia viral oncogene homolog 1 (ABL1) gene on chromosome 9, resulting in constitutive expression of a BCR-ABL1 fusion protein(1, 2). Imatinib, a small molecule tyrosine kinase inhibitor that binds to and sterically interferes with the kinase domain of BCR-ABL1, effectively halts its constitutive and oncogenic cell cycle signaling activity. Kinase inhibition, as typified by imatinib(3), now forms a major part of the therapeutic options in molecularly-based subtypes of advanced lung, breast and medullary thyroid cancers, melanoma, gastrointestinal stromal tumors, and haematological malignancies such as CML. Based on high rates of oncogenic activating BRAF mutations, current kinase inhibitorbased therapy in melanoma is centered upon targets within the MAPK signaling pathway. Due to the importance of MAPK signaling in virtually all tissues of the body, effects of these inhibitors on non-malignant cells has become a field of intense research. The MAPK pathway is also known to be an important mitogenic signaling cascade in leukocytes, and its role in myeloproliferative disease is being increasingly established however the precise effects of MAPK inhibitors are highly dependent on the specific clinical context in which these agents are used. MEK inhibition has been demonstrated to rapidly correct aberrant myeloproliferative activity seen in murine models of both chronic and juvenile myelomonocytic leukemia, relating to MAPK hyperactivity induced by an activating KRAS mutation(4) or inactivation of the NF1 tumour suppressor gene(5). Other studies have revealed that RAS-driven MAPKResearch. on April 12, 2017. © 2015 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 22, 2015; DOI: 10.1158/1078-0432.CCR-15-0393 ANDREWS et al. CLINICAL RESPONSE OF CML TO MEK INHIBITION 6 6 pathway dependence may be inadvertently intensified by BCR-ABL kinase inhibitors, leading to a synergistic anti-leukemic effect when used in combination with a MEK inhibitor(6). Similarly, a case of accelerated RAS-mutant leukemia was reported in a patient with metastatic melanoma receiving treatment with a BRAF inhibitor (7). We report the case of a patient with BRAF V600E metastatic malignant melanoma who was treated with a combination of the V600-mutation-specific BRAF inhibitor dabrafenib, and the MEK1/2 inhibitor trametinib. Incidentally, he had a synchronous diagnosis of t(9;22) positive CML with an initial circulating leukocyte count of >100 x10/L. Despite not receiving BCR-ABL targeted kinase inhibitor therapy, he achieved hematologic complete remission while on dabrafenib and trametinib treatment. Correlative ex vivo and in vitro analyses confirmed a dominant effect of the MEK inhibitor rather than the BRAF inhibitor on the patient’s leukocytes, mediated by a relatively selective reduction in cell number and MAPK signaling within precursor (primitive) cell populations and reduced colony forming potential. We also demonstrate that in CML cells, in contrast to several solid tumour types, the addition of a MEK inhibitor is able to overcome the paradoxical MAPK activation induced by BRAF inhibition. These findings suggest that further investigation of the role of MAPK pathway inhibition in de novo CML in a clinical setting is warranted. MATERIALS AND METHODS Patient Details The patient was enrolled in a phase I/II clinical trial of dabrafenib and trametinib in advanced melanoma (BRF113220, GSK, ClinicalTrials.gov number NCT01072175)(8), approved by the institutional review board of Austin Health, Research. on April 12, 2017. © 2015 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 22, 2015; DOI: 10.1158/1078-0432.CCR-15-0393 ANDREWS et al. CLINICAL RESPONSE OF CML TO MEK INHIBITION 7 7 Australia (Austin Health Human Research Ethics Committee, project number H2011/03955). All patient-derived tissue/blood samples were provided voluntarily under the auspices of the Cancer Biobanking and Research protocol approved by the institutional review board of Austin Health, Australia (H2012/04446), to which the patient had previously consented. Blood samples were collected at multiple timepoints prior to, during, and after the patient’s period of treatment with kinase inhibitors for metastatic melanoma. Baseline leukocyte samples were also obtained from peripheral blood leukapheresis. Clinical follow-up data was as per trial protocol and standard clinical care. Methods Hematologic Monitoring Differential cell counts and blood films were obtained from diagnostic blood samples collected and analysed through the local pathology service provider (Austin Pathology). Cytogenetic analyses were performed by the Victorian Cytogenetics Service, and BCR-ABL1 counts performed at the Royal Melbourne Hospital (Parkville, Victoria, Australia).