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Clinical variables associated with ecotropic viral integration site 1 (EVI1) translocations were evaluated in 42 consecutive chronic myeloid leukemia (CML) patients in myeloid blast crisis (MBC). Translocations were confirmed with fluorescence in situ hybridization, and Western blot analysis demonstrated EVI1 expression. Translocations of EVI1 were present in 3 of 24 (12%) patients whose disease evolved MBC before tyrosine kinase inhibitor (TKI) exposure, and 7 of 18 (39%) patients who had received one or more TKIs. Univariate analysis showed that prior TKI therapy was the only clinical variable that was significantly associated with EVI1 translocation (P = 0.047). TKI-resistant BCR-ABL1 mutations were present in 71% of MBC patients with EVI1 translocations at the time of disease progression. These observations suggest that EVI1 overexpression collaborates with BCR-ABL1 in the evolution of TKI-resistant MBC. Inhibition of c-ABL kinase-mediated DNA double-strand repair by TKIs may predispose to EVI1 translocation in this setting.

SummaryThe BCR-ABL inhibitor dasatinib achieves clinical remissions in chronic myeloid leukemia (CML) patients using a dosing schedule that achieves potent but transient BCR-ABL inhibition. In vitro, transient potent BCR-ABL inhibition with either dasatinib or imatinib is cytotoxic to CML cell lines, as is transient potent EGFR inhibition with erlotinib in a lung cancer cell line. Cytotoxicity correlates with the magnitude as well as the duration of kinase inhibition. Moreover, cytotoxicity with transient potent target inhibition is equivalent to prolonged target inhibition and in both cases is associated with BIM activation and rescued by BCL-2 overexpression. In CML patients receiving dasatinib once daily, response correlates with the magnitude of BCR-ABL kinase inhibition, thereby demonstrating the potential clinical utility of intermittent potent kinase inhibitor therapy.

Molecularly targeted kinase inhibitor cancer therapies are currently administered sequentially rather than simultaneously. We addressed the potential long-term impact of this strategy in patients with chronic myelogenous leukemia (CML), which is driven by the fusion oncogene BCR-ABL. Analysis of BCR-ABL genotypes in CML patients who relapsed after sequential treatment with the ABL inhibitors imatinib and dasatinib revealed evolving resistant BCR-ABL kinase domain mutations in all cases. Twelve patients relapsed with the pan-resistant T315I mutation, whereas 6 patients developed novel BCR-ABL mutations predicted to retain sensitivity to imatinib based on in vitro studies. Three of these patients were retreated with imatinib (or the chemically related compound nilotinib) and responded; however, selection for compound mutants (2 or 3 BCR-ABL mutations in the same molecule) can substantially limit the potential effectiveness of retreating patients with inhibitors that have previously failed. Furthermore, drug-resistant mutations, when compounded, can increase oncogenic potency relative to the component mutants in transformation assays. The Aurora kinase inhibitor VX-680, currently under clinical evaluation based on its activity against the T315I mutation, is also effective against the other commonly detected dasatinib-resistant mutation in our analysis, V299L. Our findings demonstrate the potential hazards of sequential kinase inhibitor therapy and suggest a role for a combination of ABL kinase inhibitors, perhaps including VX-680, to prevent the outgrowth of cells harboring drug-resistant BCR-ABL mutations.

Through sequencing analysis of blood or bone marrow samples from patients with chronic myeloid leukemia, we identified BCR-ABL kinase domain mutations in 29 of 32 patients whose disease relapsed after an initial response to the tyrosine kinase inhibitor imatinib. Fifteen different amino acid substitutions affecting 13 residues in the kinase domain were found. Mutations fell into two groups—those that alter amino acids that directly contact imatinib and those postulated to prevent BCR-ABL from achieving the inactive conformational state required for imatinib binding. Distinct mutations conferred varying degrees of imatinib resistance. Mutations detected in a subset of patients with stable chronic phase disease correlated with subsequent disease progression. Multiple independent mutant clones were detected in a subset of relapsed cases. Our data support a clonal selection model of preexisting BCR-ABL mutations that confer imatinib resistance.

The Abl tyrosine kinase inhibitor STI-571 is effective therapy for stable phase chronic myeloid leukemia (CML) patients, but the majority of CML blast-crisis patients that respond to STI-571 relapse because of reactivation of Bcr-Abl signaling. Mutations of Thr-315 in the Abl kinase domain to Ile (T315I) were previously described in STI-571-resistant patients and likely cause resistance from steric interference with drug binding. Here we identify mutations of Tyr-253 in the nucleotide-binding (P) loop of the Abl kinase domain to Phe or His in patients with advanced CML and acquired STI-571 resistance. Bcr-Abl Y253F demonstrated intermediate resistance to STI-571 in vitro and in vivo when compared with Bcr-Abl T315I. The response of Abl proteins to STI-571 was influenced by the regulatory state of the kinase and by tyrosine phosphorylation. The sensitivity of purified c-Abl to STI-571 was increased by a dysregulating mutation (P112L) in the Src homology 3 domain of Abl but decreased by phosphorylation at the regulatory Tyr-393. In contrast, the Y253F mutation dysregulated c-Abl and conferred intrinsic but not absolute resistance to STI-571 that was independent of Tyr-393 phosphorylation. The Abl P-loop is a second target for mutations that confer resistance to STI-571 in advanced CML, and the Y253F mutation may impair the induced-fit interaction of STI-571 with the Abl catalytic domain rather than sterically blocking binding of the drug. Because clinical resistance induced by the Y253F mutation might be overcome by dose escalation of STI-571, molecular genotyping of STI-571-resistant patients may provide information useful for rational therapeutic management.

Survival rates for patients with muscle-invasive bladder cancer have not improved in the past 20 years, and new therapies are imperative. Intratumor heterogeneity can complicate molecular profiling attempts to optimize therapy for cancers harboring several actionable tumor subclones. To develop personalized treatment strategies, there is a need for assays to measure intratumor heterogeneity in bladder cancers. We conducted a pilot study of two muscle invasive high-grade transitional cell carcinoma cases. We used a comprehensive cancer panel (Thermo Fisher) covering >400 cancer genes to analyze distinct tumor loci and matched normal tissues. Based on the identified somatic mutations, we designed a bladder-specific panel to (1) validate our results with increased coverage, and (2) analyze liquid biopsy samples. Using the comprehensive cancer panel, we sequenced 6 tumor loci to an average sequencing depth of approximately 100x. We detected intratumor heterogeneity in both patients: By applying a combination of frequency-based (minor allele frequency >10%) and probabilistic (probability of difference between observed frequencies due to sampling) filters, we identified 44 credible somatic SNVs, including mutations that were not shared among all three loci. We used these SNVs to design a custom amplicon panel covering 42 SNVs across 38 genes that is suitable for highly fragmented DNA. The custom panel was used to validate the SNVs in the same tumor regions and in liquid biopsy samples from plasma and urine (approximate coverage 6,000x). In both cases, we identified private mutations reported in The Cancer Genome Atlas Urothelial Bladder Carcinoma (TCGA-BLCA) data collection, reflecting tumor evolution. Liquid biopsy samples from urine revealed all trunk mutations but only 1 out of 5 private mutations. We conclude that tumor evolution can affect distinct loci within bladder tumors, which may not be fully represented in liquid biopsy samples. These results suggest the need for analyzing multiple tumor regions to identify all actionable driver mutations. In the future, we plan to apply our assay to additional foci and patients in order to identify optimal bladder tumor sampling strategies. Citation Format: Katherin Patsch, Naim Matasci, Anjana Soundararajan, John Nicoll, Jonathan Katz, Antonio Sanchez, Erika Feierstein, Christina Van Loy, Zhao Xu, David B. Agus, Mitchell E. Gross, Daniel Ruderman. Bladder cancer tumor heterogeneity: development of a system-level mutation assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3934. doi:10.1158/1538-7445.AM2017-3934

IL-16 is synthesized as a precursor molecule of 68 kDa (pro-IL-16) that is processed by caspase-3, a member of the IL-1 converting enzyme (ICE) family. This cleavage results in a 13-kDa carboxy terminal peptide, which constitutes the bioactive secreted form of IL-16. We have previously reported constitutive IL-16 mRNA expression and pro-IL-16 protein in CD4+ and CD8+ T cells. Although bioactive IL-16 protein is present in unstimulated CD8+ T cells, there is no bioactive IL-16 present in CD4+ T cells. Along these lines, unstimulated CD8+ T cells contain active caspase-3. In the current studies we investigated the regulation of IL-16 protein and mRNA expression in CD4+ T cells and determined the kinetics of secretion following stimulation of the TCR. CD4+ T cells release IL-16 protein following antigenic stimulation, and this release is accelerated in time by costimulation via CD28. However, CD3/CD28 costimulation did not alter IL-16 mRNA appearance or stability in either CD4+ or CD8+ T cells. The secretion of bioactive IL-16 from CD4+ T cells correlated with the appearance of cleavage of pro-caspase-3 into its 20-kDa active form. Thus, resting CD8+ T cells contain active caspase-3 that is capable of cleaving pro-IL-16, whereas CD4+ T cells require activation for the appearance of active caspase-3. The mechanism of release or secretion of bioactive IL-16 is currently unknown, but does not correlate with cellular apoptosis.

Progression of chronic myelogenous leukemia (CML) to accelerated (AP) and blast phase (BP) is because of secondary molecular events, as well as additional cytogenetic abnormalities. On the basis of the detection of JAK2, CBL, CBLB, TET2, ASXL1, and IDH1/2 mutations in myelodysplastic/myeloproliferative neoplasms, we hypothesized that they may also contribute to progression in CML. We screened these genes for mutations in 54 cases with CML (14 with chronic phase, 14 with AP, 20 with myeloid, and 6 with nonmyeloid BP). We identified 1 CBLB and 2 TET2 mutations in AP, and 1 CBL, 1 CBLB, 4 TET2, 2 ASXL1, and 2 IDH family mutations in myeloid BP. However, none of these mutations were found in chronic phase. No cases with JAK2V617F mutations were found. In 2 cases, TET2 mutations were found concomitant with CBLB mutations. By single nucleotide polymorphism arrays, uniparental disomy on chromosome 5q, 8q, 11p, and 17p was found in AP and BP but not involving 4q24 (TET2) or 11q23 (CBL). Microdeletions on chromosomes 17q11.2 and 21q22.12 involved tumor associated genes NF1 and RUNX1, respectively. Our results indicate that CBL family, TET2, ASXL1, and IDH family mutations and additional cryptic karyotypic abnormalities can occur in advanced phase CML.

Mammalian microRNAs are emerging as key regulators of the development and function of the immune system. Here, we report a strong but transient induction of miR-155 in mouse bone marrow after injection of bacterial lipopolysaccharide (LPS) correlated with granulocyte/monocyte (GM) expansion. Demonstrating the sufficiency of miR-155 to drive GM expansion, enforced expression in mouse bone marrow cells caused GM proliferation in a manner reminiscent of LPS treatment. However, the miR-155–induced GM populations displayed pathological features characteristic of myeloid neoplasia. Of possible relevance to human disease, miR-155 was found to be overexpressed in the bone marrow of patients with certain subtypes of acute myeloid leukemia (AML). Furthermore, miR-155 repressed a subset of genes implicated in hematopoietic development and disease. These data implicate miR-155 as a contributor to physiological GM expansion during inflammation and to certain pathological features associated with AML, emphasizing the importance of proper miR-155 regulation in developing myeloid cells during times of inflammatory stress.

Background The BCR-ABL tyrosine kinase inhibitor imatinib is effective in Philadelphia chromosome–positive (Ph-positive) leukemias, but relapse occurs, mainly as a result of the outgrowth of leukemic subclones with imatinib-resistant BCR-ABL mutations. We evaluated dasatinib, a BCR-ABL inhibitor that targets most imatinib-resistant BCRABL mutations, in patients with chronic myelogenous leukemia (CML) or Ph-positive acute lymphoblastic leukemia (ALL). Methods Patients with various phases of CML or with Ph-positive ALL who could not tolerate or were resistant to imatinib were enrolled in a phase 1 dose-escalation study. Dasatinib (15 to 240 mg per day) was administered orally in four-week treatment cycles, once or twice daily. Results A complete hematologic response was achieved in 37 of 40 patients with chronicphase CML, and major hematologic responses were seen in 31 of 44 patients with accelerated-phase CML, CML with blast crisis, or Ph-positive ALL. In these two phases, the rates of major cytogenetic response were 45 percent and 25 percent, respectively. Responses were maintained in 95 percent of patients with chronic-phase disease and in 82 percent of patients with accelerated-phase disease, with a median follow-up more than 12 months and 5 months, respectively. Nearly all patients with lymphoid blast crisis and Ph-positive ALL had a relapse within six months. Responses occurred among all BCR-ABL genotypes, with the exception of the T315I mutation, which confers resistance to both dasatinib and imatinib in vitro. Myelosuppression was common but not dose-limiting. Conclusions Dasatinib induces hematologic and cytogenetic responses in patients with CML or Ph-positive ALL who cannot tolerate or are resistant to imatinib. (ClinicalTrials.gov number, NCT00064233.)

IL-16 is a proinflammatory cytokine implicated in the pathogenesis of asthma and other conditions characterized by recruitment of CD4+ T cells to sites of disease. It is postulated that CD4 is an IL-16 receptor, although other receptors or coreceptors may exist. Among several known functions, IL-16 is a chemoattractant factor for CD4+ T cells and it inhibits MLR. We previously reported that an oligopeptide corresponding to the 16 C-terminal residues of human IL-16 inhibits chemoattractant activity. To identify functional domains with greater precision, shorter oligonucleotides containing native or mutated C-terminal IL-16 sequences were tested for IL-16 inhibition. Within the 16 C-terminal residues, the minimal peptide RRKS (corresponding to Arg106 to Ser109) was shown to mediate inhibition of IL-16 chemoattractant activity. Inhibition was lost when either arginine was substituted with alanine. Point mutations in IL-16 revealed that Arg107 is critical for chemoattractant activity, but MLR inhibition was unaffected by mutation of Arg107 or even deletion of the C-terminal tail through Arg106. Deletion of 12 or 22 N-terminal residues of IL-16 had no impact on chemoattractant activity, but MLR inhibition was reduced. Deletion of 16 C-terminal plus 12 N-terminal residues abolished both chemoattractant and MLR-inhibitory activity of IL-16. These data indicate that receptor interactions with IL-16 that activate T cell migration are not identical with those required for MLR inhibition, and suggest that both N-terminal and C-terminal domains in IL-16 participate in receptor binding or activation.

Abstract 3396 Chronic myeloid leukemia (CML) is characterized by the BCR/ABL fusion gene. However, secondary molecular events leading to accelerated (AP) or blast phase (BP) have not been sufficiently clarified. We hypothesized that, in analogy to other MDS/MPN or MPN, TET2, ASXL1, CBL and IDH family mutations may also occur in CML and contribute as secondary events leading to progression to AP or BP. Similarly, higher resolution of cytogenetic testing by single nucleotide polymorphism array (SNP-A)-based karyotyping may reveal additional chromosomal abnormalities associated with stepwise progression. This study is focused on the combined analysis of chromosomal lesions and mutations associated with AP, BP and Philadelphia chromosome (Ph) positive acute lymphoblastic leukemia (ALL) and the association of these defects with clinical features. We screened TET2, ASXL1, CBL and IDH for mutations in AP (N=14) and BP (N=26) and Ph+ ALL (N=9). Chronic phase (CP) (N=14) and Ph negative ALL (N=9) served as controls. We identified 3 CBL family (9%), 7 TET2 (21%), 2 ASXL1 (6%) and 2 IDH family (6%) mutations in patients with AP and myeloid BP. Subsequently, we also detected a TET2 mutation in a case of Ph+ ALL. None of these mutations were found in patients with CP or Ph negative ALL. We also performed SNP-A-based karyotyping and only included lesions which did not overlap with copy number variations (CNVs) or germ line regions of homozygosity present in any of the controls. 23 gains, 21 losses and 4 regions of somatic UPD lesions were identified. By SNP-A, additional copy number abnormalities, including microdeletions were found in 67% and 50% of patients with AP and BP, respectively. Recurrent lesions were detected on chromosome 1, 8, 9, 17 and 22. Microdeletions on chromosome 17 and 21 involved tumor associated genes NF1 and RUNX1. Deletions flanking the ABL1 and BCR genes were observed in 3 cases with der(22)t(9;22) or der(9)t(9;22) by metaphase cytogenetics. Gains including 1q25.3q41, chromosome 8 and 17q24.3 were found in 3 cases. Regions of UPD included UPD5q, 8q, 11p and 17q but no UPD involving 11q (CBL) and 4q (TET2) regions were found confirming heterozygous nature of the corresponding mutations. Newly detected molecular lesions associated with AP and BP may change the biology and thereby clinical features of affected cases. Overall survival of patients with mutations did not differ from those without mutations. Of note is that BCR/ABL1 kinase domain mutations were detected in 9/10 patients with imatinib resistance. In these 9 cases, 3 TET2 and 2 CBLB mutations were detected (but no mutations in the other genes). In an imatinib-resistant patient without BCR/ABL1 kinase domain mutation, CBL mutation was present. In the patients with TET2 mutations, additional chromosomal lesions were found by SNP-A, significantly more frequently when compared with WT cases (P=0.017). Of the 9 TET2 variants in 8 cases, 7 (78%) were missense substitutions, 1 (11%) was frame shift and 1 (11%) produced a stop codon and were located within the N-terminus as well as in a conserved DSBH 2OG-Fe(II)-dependent dioxygenase domain. The presence of nonsense and frameshift mutations suggests that mutated lesions result in inactivation, consistent with putative tumor suppressor functions, while heterozygous mutations indicate that the wild type allele is not completely protective. Since no TET2 mutations were identified in chronic phase CML, these mutations might represent an additional pathogenic event and contribute to progression. In 3 cases we observed a combination of 2 mutations. Coincidence of CBLB and TET2 mutations in 2 cases suggests that these might cooperate in the evolution of advanced phase of CML. We also found a combination of IDH1 and ASXL1 mutations in a patient with BP, suggesting that both mutations contribute to clonal advantage. In conclusion, while CBL family, ASXL1 and IDH family mutations as well a additional unbalanced chromosomal abnormalities not seen by metaphase cytogenetics can occur in myeloid type advanced phase CML, TET2 mutations were identified in Ph+ ALL, as well as myeloid BP and AP. These mutations likely represent secondary lesions which contribute to either disease progression or more aggressive features and commonly occur in association with imatinib-resistant BCR/ABL mutations. Disclosures: No relevant conflicts of interest to declare.

Patients with chronic myeloid leukemia (CML) are experiencing prolonged survival due to successful therapy with tyrosine kinase inhibitors. However, some CML patients who have achieved longstanding remissions with these agents harbor clonal cytogenetic abnormalities in their Philadelphia chromosome negative (Ph-) bone marrow cells. Because CML patients in remission often have peripheral blood count abnormalities, including cytopenias, we investigated whether these patients may have developed myelodysplastic syndrome (MDS) within the Ph- cell population. Bone marrow samples from 26 CML patients who had achieved a major cytogenetic remission (MCyR) with tyrosine kinase inhibitor therapy between 2 and 15 years after diagnosis were evaluated; 6 patients had advanced disease prior to their last therapy, 20 were in chronic phase. At the time of evaluation, 2 of the patients were receiving imatinib, 23 dasatinib, and 1 PHA739358. At least one peripheral blood lineage was abnormal in 21 patients, of whom 7 had pancytopenia. Routine metaphase cytogenetics (MC) revealed a persistent clonal chromosomal abnormality in 10% of the Ph- metaphases in 5 patients (+8 in 2, −7 in 2, and 20q- in 1). We hypothesized that clonal hematopoiesis might exist in additional patients and applied single nucleotide array (SNP-A) based karyotyping and X-linked human androgen receptor (HUMARA) clonality assay to further delineate the nature of the hematopoietic defect in these patients. HUMARA was performed on bone marrow samples and germ-line DNA from peripheral blood T lymphocytes of the female patients. Clonality, as assessed by skewing of X-chromosome inactivation in bone marrow cells compared to germline control cells, could not be demonstrated in the12 female patients. SNP-A karyptyping using 250K Affymetrix SNP array confirmed the known cytogenetic abnormalities. Several microdeletions were found, but comparison with purified T lymphocytes demonstrated that these “lesions” represented germ line-encoded copy number variants. However, SNP-A karyotyping revealed the presence of uniparental disomy (UPD) involving chromosome 17(p12-pter) in bone marrow, but not germ line cells, from one male patient with normal karyotype by routine MC. In the context of secondary AML, del17p or UPD17 have been observed always in the presence of del7/q and 5q and were associated with poor prognosis. However, in our patient UPD17 occurred as a sole defect. Because in our studies in AML, UPD of chromosome 17p was found in association with p53 mutations, genomic sequencing of this gene was performed. A 5 bp deletion destroying the splice acceptor region of exon 6 was identified in bone marrow cells from this patient. Alternative splicing leading to loss of exon 6 was predicted to result in a frame shift and premature introduction of a stop codon. These methods revealed clonal hematopoiesis in the Ph- bone marrow cells of 6/26 patients with longstanding CML in remission from tyrosine kinase inhibitors and persistent peripheral blood abnormalities. The approaches used here probably underestimate the frequency of this condition, as oligoclonal populations may be present in numbers below the limit of assay sensitivity. The Ph- clonal bone marrow populations have cytogenetic and molecular features in common with MDS. After a median follow up of two years, one patient with monosomy 7 developed acute myeloid leukemia, but longer follow up will be required to determine the natural history of the Ph- clonal disorders.

Recent discoveries have implicated microRNAs, which are small 22–24 nucleotide long RNA molecules, as important regulators of many cellular processes, including differentiation and development. The microRNA-155 (miR-155) is known to be overexpressed in subsets of B-cell neoplasms and is thought to be important in the activation and function of B-lymphocytes. Here, we show that miR-155 is signficantly overexpressed in human acute myeloid leukemia and that its overexpression is most consistently seen in acute myelomonocytic leukemia. These findings led us to investigate the effects of overexpression of miR-155 in hematopoietic cells. By utilizing retroviral infection and transfer of murine bone marrow, we introduced miR-155 overexpressing hematopoietic stem cells into lethally irradiated recipient mice. Following reconstitution of hematopoietic organs, the mice demonstrated a profound myeloproliferative condition in the bone marrow characterized by replacement of the marrow by proliferating and dysplastic myelomonocytic cells. In addition, extramedullary hematopoiesis was observed in the spleen and examination of the peripheral blood revealed anemia and thrombocytopenia. To begin to explore the mechanisms whereby miR-155 overexpression results in this myeloproliferative phenotype, we utilized computational methods to identify targets predicted to be regulated by miR-155. This revealed several genes that have previously been implicated in myeloid development and neoplasia, which were confirmed to be targets of miR-155 by reverse-transcription/quantitative polymerase chain reaction (RT/QPCR) and by downregulation of luciferase protein upon fusion of the luc gene with the respective 3′ untranslated regions. These studies show a hitherto uncharacterized role of miR-155 in myeloid development and proliferation. Importantly, these findings lay the groundwork for understanding the complex regulatory networks underlying myeloid development in the context of microRNAs, and may point to new therapeutic opportunities in the treatment of myeloid malignancies.

A critical question in the targeted therapy era relates to whether treatment outcomes will be optimized by sequential or combinatorial use of targeted agents. Selection for CML cells with BCR-ABL kinase domain mutations is the main mechanism responsible for loss of response to imatinib. Dasatinib is an ABL tyrosine kinase inhibitor that has activity against nearly all imatinib-resistant mutations and is approved for the treatment of imatinib-resistant and -intolerant BCR-ABL-associated leukemias. Acquired clinical resistance to sequential use of dasatinib following imatinib failure has been observed. We analyzed the BCR-ABL kinase domain at the time of relapse in 15 patients who lost an initial response to dasatinib, and found evolution of a total of three new mutations at the time of relapse in all cases. The highly resistant BCR-ABL/T315I mutation was detected in 11 cases. The four remaining cases were associated with the evolution of novel mutations (V299L, 3 cases; T315A, 1 case). V299L was also detected in a fourth case that had also evolved T315I. These three dasatinib-resistant mutations were part of a small number of amino acid substitutions previously isolated in a preclinical mutagenesis screen for dasatinib resistance-conferring BCR-ABL mutations. While the T315I mutation is highly resistant to imatinib, V299L and T315A retain sensitivity to imatinib in vitro and have not been previously described in imatinib-resistant cases, raising the potential utility of imatinib rechallenge in select dasaitinib-resistant cases. A significant finding of our studies is the evolution of five unique “compound” mutations (i.e. greater than one mutation on a DNA strand) in the BCR-ABL kinase domain of patients treated sequentially with imatinib and dasatinib. It is noteworthy that although the imatinib-sensitive V299L and T315A mutations evolved in five cases, they were detected in the context of a pre-existing imatinib-resistant mutation in three of these cases, and these cases are therefore unlikely to respond to rechallenge with IM. The T315A mutation was detected in the context of 2 pre-existing IM-resistant mutations (M244V/L364I). Interestingly, in bone marrow transformation assays, the clinically-identified dasatinib-resistant M244V/L364I/T315A mutation was more potently oncogenic than non-mutated BCR-ABL, in contrast to the baseline imatinib resistant M244V/L364I, which like T315A in isolation, was less potent than native BCR-ABL Our studies of CML cases resistant to sequential kinase inhibitor therapy reinforce BCR-ABL kinase domain mutation as the predominant mechanism of resistance to kinase inhibitor therapy, and provide evidence that compound mutations acquired as a result of sequential therapy can not only limit further therapeutic options, but also create more biologically aggressive isoforms of BCR-ABL. Together, these findings provide a strong rationale for early treatment of CML with combinations of kinase inhibitors that have the capacity to collectively prevent selection of resistant kinase domain mutations.

Dasatinib (Sprycel; formerly BMS-354825) is an oral multi-targeted SRC/ABL tyrosine kinase inhibitor that is approximately 325-fold more potent than imatinib, and has been recently approved by the US FDA for the treatment of imatinib-resistant and -intolerant Philadelphia chromosome-associated leukemias. In contrast to other tyrosine kinase inhibitors, pharmacokinetic analyses reveal that dasatinib has a short half-life (5–6 hours), with near-complete loss of BCR-ABL kinase activity inhibition eight hours after drug administration in patients with CML. Remarkably, patients treated with dasatinib as infrequently as once daily, five days per week, have achieved complete cytogenetic remission (CCyR). We tested transient BCR-ABL inhibition in vitro by assessing the sensitivity of the CML cell line K562 to short-term exposure of dasatinib. After as short as a 20-minute exposure to a clinically-relevant dasatinib concentration (100 nM), the majority of cells undergo apoptosis when analyzed after 48 hours, whereas a clinically-relevant concentration of imatinib (5 uM) failed to result in substantial cytotoxicity under these conditions. When higher concentrations (>=12.5 uM) of imatinib were used that correct for differences in potency between imatinib and dasatinib, cytotoxicity at 48 hours after a 20-minute exposure was similar to that observed with dasatinib. These results exclude SRC family inhibition as a potential mediator of this effect, and were reproducible in a second CML cell line, KU-812. Importantly, cytotoxicity was not observed in BCR-ABL-negative leukemia cell lines under these conditions. Signiificantly, similar phenomenon was observed when an erlotinib-sensitive non-small cell lung cancer was exposed to high concentrations of erlotinib for 20 minutes. We assessed the degree of BCR-ABL kinase inhibition achieved in 20 patients with chronic phase CML who were treated with dasatinib once daily as part of a phase I clinical trial by quantifying the ratio of phospho-CRKL to CRKL (a BCR-ABL substrate) achieved in PBMCs harvested four hours after the first dose of dasatinib. We found a strong correlation between the magnitude of BCR-ABL kinase inhibition and the depth of response achieved. Notably, CCyR was achieved exclusively in four patients who experienced the deepest inhibition of BCR-ABL kinase activity activity. The current medical management of CML involves assessing the degree of cytogenetic response after 6–12 months of imatinib therapy before considering dose modifications of imatinib. While our findings will need to be validated prospectively in larger cohorts of patients, they suggest that it may be feasible to make early dose modifications based upon the degree of target inhibition following the initial dose of dasatinib, and thus maximize the likelihood of clinical benefit. Together, these findings have potentially significant implications not only for the optimal clinical management of CML patients, but also for the rational development of small molecule inhibitors of other cancer-associated tyrosine kinases, as well as our global understanding of cancer cell biology.

Selection for CML cells with BCR-ABL kinase domain mutations represents the predominant molecular mechanism responsible for loss of response to imatinib. Similarly, we have found acquired resistance to dasatinib to be associated with kinase domain mutations in 100% of cases (n=15). However, unlike the multitude of imatinib resistant mutations, which to a large extent occur at non-contact residues and destabilize the inactive confirmation to which imatinib binds, only two mutations appear to be responsible for nearly all cases of dasatinib resistance, T315I and V299L (Shah et al, submitted, ASH 2006). Both of these mutations occur at critical contact residues between the ABL kinase domain and dasatinib. Successful treatment of dasatinib-resistant cases will therefore require strategies to successfully eliminate cells that harbor these mutations. Use of a combination of kinase inhibitors with the ability to collectively suppress all BCR-ABL kinase domain mutants would be predicted to lead to profoundly minimize disease resistance and relapse on targeted therapy. Although the combination of imatinib and dasatinib may prevent selection of the dasatinib-resistant V299L mutation, these agents share many targets, and their combination may therefore result in substantial toxicity. Moreover, the combination of imatinib and dasatinib is not predicted to effectively inhibit the growth of cells harboring BCR-ABL/T315I. We previously have shown that the Aurora kinase inhibitor VX-680 can bind to the ABL kinase domain and inhibit the kinase activity of the T315I mutation at low micromolar concentration. VX-680 is showing early signs of efficacy in CML cases associated with the T315I mutation. Interestingly, the co-crystal structure of VX-680 reveals that V299 is one of 14 contact residues within the ABL kinase domain. Substitution of leucine at this residue might therefore be expected to diminish the potential affinity of VX-680 for BCR-ABL/V299L. However, analysis of the amino acid sequence of Aurora-A revealed divergence from native BCR-ABL at this the corresponding amino acid position due to the presence of a leucine in Aurora-A. BCR-ABL/V299L is therefore a mimetic of Aurora-A, and as a result, predicted to retain sensitivity to VX-680. We therefore assessed VX-680 for its ability to inhibit the kinase activity of BCR-ABL/V299L in Ba/F3 cells and found effective inhibition of the kinase activity at low micromolar concentration. Consistent with predictions based upon structural considerations, the V299L mutation is somewhat more sensitive to VX-680 than BCR-ABL/T315I. We confirmed these results in an analysis of primary human PBMCs obtained from a dasatinib-resistant patient who had evolved the V299L mutation on therapy. Our findings suggest that early combination therapy with two kinase inhibitors, dasatinib and VX-680, may successfully suppress resistant disease by collectively eliminating BCR-ABL kinase domain mutation as a mechanism of resistance, and thereby achieve effective long-term disease control in the vast majority of patients.

Acquired imatinib-resistance in Ph-positive leukemia is frequently associated with mutations in the BCR-ABL kinase domain. Dasatinib is more potent than imatinib for inhibiting BCR-ABL activity and has preclinical efficacy against imatinib resistant mutations tested so far, except for T315I. We report the molecular analysis of imatinib resistant/intolerant patients treated at UCLA in a Phase I dose escalation trial of dasatinib. We sought to determine whether patients achieve significant reductions in the BCR-ABL level as measured by quantitative PCR (RQ-PCR); the effect of dasatinib on mutant BCR-ABL clones in-vivo; and the contribution of mutations to progression. Patients with accelerated phase or blast crisis CML, or Ph+ALL (AP/BC, n=14) were treated for a median of 5 months (range 1 to 11) and late (median disease duration 8 years) chronic phase CML patients (CP, n=19) for a median of 12 months (range 3 to 21). Patients were tested 2 to 17 times by RQ-PCR (267 analyses) and direct sequencing (167 analyses). Prior to commencing dasatinib, mutations were detected in resistant patients only (8 AP/BC and 15 CP). For our molecular analysis a ≥2-log reduction of BCR-ABL below the standardized baseline was considered significant. This level approximates to a complete cytogenetic response. A 3-log reduction defines a major molecular response (MMR), which is associated with a high progression free survival in imatinib treated patients. Overall, 6 of 14 (43%) AP/BC and 7 of 19 (37%) CP patients achieved ≥2-log reductions (MMR in 4 (29%) and 4 (21%) patients respectively). The response was maintained in 2 of 6 AP/BC and 6 of 7 CP patients achieving ≥2 log reductions. The table classifies the molecular response according to the detection of a baseline mutation. Molecular response ≥2 log BCR-ABL reduction intolerant and no baseline mutant resistant and no baseline mutant resistant and baseline mutant CP 2/3 (67%) 1/1 (100%) 4/15 (27%) AP/BC 1/1 (100%) 3/5 (60%) 2/8 (25%) Overall 3/4 (75%) 4/6 (67%) 6/23 (26%) Mutations were detected at last analysis in all 23 patients with baseline mutations. The sensitivity of the direct sequencing technique is approximately 20%. The same mutation that was present at baseline was present in 21 patients and 5 of these patients had an additional mutation. The remaining 2 patients had different mutations, one being F317I (CP). This mutation has not been reported in imatinib resistant patients to our knowledge. Mutations were present in all patients who progressed (1 CP, 7 AP/BC). Six of these patients had T315I that was detected at baseline (3) or evolved during therapy (3). T315I evolved in 3 other patients who have not progressed (1CP, 2 AP/BC). Overall, the T315I mutation evolved in 6 patients and was accompanied by significant rises in BCR-ABL in all patients of 2.5 to 185-fold. In conclusion 39% of all patients achieved significant molecular responses, with major molecular responses in 24% overall. The majority of CP patients maintained the molecular response. Baseline mutations remained detectable in almost all patients including those with significant BCR-ABL reductions. The mutation that became detectable most frequently during dasatinib therapy was T315I and non-mutation relapse was rare. A focus of future trials may be on managing the remaining resistant mutations.

Imatinib mesylate resistance in CML and Ph+ ALL is frequently associated with BCR-ABL mutations that interfere with the ability of imatinib to inhibit BCR-ABL. Dasatinib (BMS-354825) is a novel, oral, multi-targeted kinase inhibitor, which targets BCR-ABL and SRC kinases. Dasatinib is 325-fold more potent versus imatinib in cells transduced with wild-type BCR-ABL, and has demonstrated preclinical activity against 18 of 19 imatinib-resistant BCR-ABL mutants (O’Hare, et al. Cancer Res65(11):4500–5, 2005; Shah et al, Science, 305:399–401, 2004). Here we present an update of a Phase I dose-escalating study, initiated in November 2003, of dasatinib in imatinib-resistant/intolerant patients with CML in late chronic phase (CP) or advanced disease (accelerated phase [AP], myeloid blast crisis [MBC] or lymphoid blast crisis [LBC]) or with Ph+ ALL. Data are available for 84 patients (40 CP, 10 AP, 23 MBC, 11 LBC/Ph+ ALL). As of this writing, 40 CP patients, with 8 years’ median duration of CML (range: 1–17 yrs), have been treated with dasatinib (15–180 mg/day, once-daily [QD] or twice daily [BID]) for a median of 13 months. The rate of complete hematologic response (CHR) in CP is 88% (35/40). Major cytogenetic responses (MCyR) were observed in 40% (16/40), with complete CyR (CCyR) in 33% (13/40). In advanced disease, 44 patients have been treated with dasatinib (70–240 mg/day, BID) for a median of 3–7 months, depending on the cohort (see Table); 2 patients (1 MBC and 1 LBC) are not evaluable for response, but are included in the analysis of time to progression. The rate of major hematologic response (MHR) (bone marrow blasts Phase of CML/ALL Patients remaining in response at July 31, 2005 Follow-up, median (range) days CP (N=40) 36 (90%) 392 (95–588) AP (N=10) 8 (80%) 200 (39–365) MBC (N=23) 9 (39%) 144 (2–364) LBC/Ph+ ALL (N=11) 1 (9%) 77 (8–233)

Point mutations within the BCR-ABL kinase domain represent the most common mechanism of resistance to imatinib in patients with CML. Preclinical studies have shown that dasatinib (BMS-354825) is effective at inhibiting the kinase activity of imatinib-resistant BCR-ABL mutants with the notable exception of the T315I mutation, which remains highly resistant to imatinib, dasatinib, and AMN107 (Gorre et al, Science 2001; Shah et al, Science 2004; Weisberg et al, Cancer Cell, 2005). Clinical data from Phase I and II studies of dasatinib in CML confirms the in vitro findings. Each of three imatinib-resistant patients bearing the T315I mutation (CP=1; AP=2) did not achieve objective hematologic or cytogenetic responses during treatment with dasatanib on a Phase I study. Additionally, each of two phase II patients with the T315I mutation (CP=1; LBC=1) treated at UCLA showed no evidence of objective response. We have also detected the T315I mutation in each of two cases of acquired resistance in a phase II (LBC =2) study, and in seven of nine patients with acquired resistance to dasatinib in phase I and II studies (CP=1; MBC=3; LBC=2; Ph+ ALL=1). Notably, we detected a novel BCR-ABL mutation, T315A, in one of the two patients who relapsed without a detectable T315I mutation. The patient is a 53 year-old female whose chronic phase CML had progressed to myeloid blast phase while being treated with imatinib. The imatinib-resistant mutation M244V was identified prior to dasatinib treatment. The patient achieved a major hematologic response (

Background/Aims Chronic myeloid leukemia (CML) is driven by the enzymatic activity of BCR-ABL, a tyrosine kinase that is selectively inhibited by imatinib. However, a subset of patients develop resistance. BMS-354825 retains in vitro activity against 14 out of 15 imatinib-resistant forms of BCR-ABL in the nanomolar concentration range. BMS-354825 is currently undergoing analysis in a phase I clinical trial of imatinib-resistant or -intolerant CML cases. We sought to determine if a correlation exists between specific kinase domain mutation and response to BMS-354825. Methods Fifteen patients were included in the analysis. All had a diagnosis of Philadelphia chromosome-positive CML, and were either imatinib-resistant or -intolerant. Sequencing of the BCR-ABL kinase domain was performed prior to treatment. The achievement of a complete hematologic response was correlated with BCR-ABL genotype. Results Preliminary analysis suggests a strong correlation between response and absence of the T315I mutation, and no significant response in cases of the T315I mutant. These data corroborate our preclinical results, and identify the T315I mutation as a potentially major component of clinical resistance to BMS-354825. Conclusions These studies suggest an evolving role for patient-specific therapy based upon the specific genotype of the target gene in human cancers treated with the burgeoning class of small molecule kinase inhibitors, and warrant confirmatory analysis in a larger cohort of patients. Clinical Pharmacology & Therapeutics (2005) 77, P17–P17; doi: 10.1016/j.clpt.2004.11.066

Disease relapse in CML patients treated with imatinib is often associated with mutations in the BCR-ABL gene that interfere with the ability of imatinib to block BCR-ABL kinase activity. BMS-354825 is a novel, orally available, dual SRC/ABL kinase inhibitor with more than 100-fold greater potency than imatinib that has in vitro and in vivo preclinical activity against 14 of 15 imatinib resistant BCR-ABL mutants (Shah et al, Science, 305:399, 2004). Here we report the phase I clinical results of BMS-354825 in Philadelphia chromosome positive (Ph+) CML patients in chronic phase with hematologic progression or intolerance while being treated with imatinib. To date (Aug 6, 2004), 29 patients have been treated on 9 cohorts with doses ranging from 15 to 180 mg of BMS-354825 per day given in single or divided doses for 5–7 days per week, for up to 9 months. Similar to imatinib, BMS-354825 has been well tolerated in all patients, with a single episode of grade 4 thrombocytopenia as the only potential drug related adverse event. BMS-354825 is rapidly absorbed with peak concentrations achieved within 2 hours and a terminal phase half-life of about 5 hours. Serum levels well above the concentration required to block CML cell proliferation in vitro have been readily achieved without side effects. Pharmacodynamic studies demonstrate greater than 50 percent inhibition of phosphorylation of the BCR-ABL substrate CRKL and the SRC kinase Lyn, consistent with the serum concentrations observed in pharmacokinetic studies. Patients not receiving optimal clinical benefit were escalated to the next higher dose for which safety parameters were available, thereby allowing a preliminary assessment of clinical activity. To date, 26 patients (22 with imatinib resistance, 4 with imatinib intolerance; average CML duration 6.1 years) have been followed for greater than 4 weeks and are eligible for assessment of hematologic benefit. 22 patients had detectable BCR-ABL kinase domain mutations prior to starting BMS-354825. All 26 patients have been treated with doses of 35 mg per day or greater and have had clinical benefit, including 19 with complete hematologic responses (73%). Of the 7 partial responders, two subsequently had disease progression, one of whom had expansion of a CML subclone containing the imatinib-resistant T315I mutation in BCR-ABL, which also confers resistance to BMS-354825 in preclinical studies. The other 5 partial responders are now being treated with higher doses to attempt conversion to complete hematologic response. 11 of 21 patients (52%) treated for greater than 3 months have cytogenetic benefit, including 6 major (1–35% Ph+), 1 minor (36–65% Ph+) and 4 minimal (66–95% Ph+) cytogenetic responses. One patient has achieved complete cytogenetic response. Dose escalation continues, and phase II studies in chronic, accelerated and blast crisis CML are currently being initiated. These data provide compelling evidence supporting the safety and efficacy of BMS-354825 in imatinib-resistant chronic phase CML.

The success of imatinib for the treatment of chronic myeloid leukemia (CML) has created a need for a sensitive and accurate method of monitoring disease response and burden. Quantitative PCR (Q-PCR) has been previously shown to correlate well with cytogenetic response in patients treated with imatinib, whereby a one-log reduction in BCR-ABL transcript level corresponded well with attainment of a major cytogenetic response (MCyR) and a two-log reduction correlated with a complete cytogenetic response (CCyR) (Branford et al, Leukemia17:2401, 2003). BMS-354825 is a novel orally bioavailable SRC/ABL kinase inhibitor with impressive activity against imatinib-resistant BCR-ABL mutant isoforms in vitro (Shah et al, Science305:399, 2004). The compound is presently undergoing evaluation in phase I clinical trials (see Sawyers et al, Talpaz et al, abstracts submitted for this meeting). We sought to address whether cytogenetic responses in patients treated with BMS-354825 correlated with reduction in BCR-ABL transcript levels as determined by Q-PCR. Of 13 evaluable imatinib-resistant/intolerant patients with chronic phase CML treated at UCLA, four have attained a MCyR. Achievement of MCyR corresponded with a one to two-log reduction in BCR-ABL transcript as assessed by Q-PCR. In the majority of cases, a substantial reduction in BCR-ABL transcript was detected four weeks after initiation of BMS-354825. Overall, the median reduction in BCR-ABL transcript level after four weeks of therapy was 32%. Three of these patients had developed resistance to imatinib, and two harbored the common imatinib-resistant M351T mutation. Of the nine patients who have failed to achieve a MCyR, none have achieved a one log reduction in BCR-ABL transcript level. We conclude that similar to imatinib, BMS-354825-associated MCyR in chronic phase CML is highly associated with a one to two-log reduction in BCR-ABL transcript level. Furthermore, Q-PCR offers a rapid and reliable method to assess for disease response in this setting, which promises to be of significant clinical value. Although the maximal tolerated dose of BMS-354825 has yet to be determined, the compound is clearly capable of substantially reducing disease burden in patients with imatinib-resistant CML. Updated Q-PCR data from all chronic, accelerated, and blast crisis-phase patients on study at UCLA will be presented.

BMS-354825 is a novel, orally available, dual SRC/ABL kinase inhibitor with 100-fold greater potency to inhibit BCR-ABL in vitro than imatinib and has in vitro and in vivo preclinical activity against 14 of 15 imatinib resistant BCR-ABL mutants (Shah et al, Science, 305:399, 2004). Here we report the phase I clinical results of BMS-354825 in Philadelphia chromosome positive accelerated phase (AP) and blast phase (BP) CML patients who had hematologic progression or intolerance while being treated with imatinib. As of Aug 6, 2004, 17 patients (6 with AP; 11 with BP) have been treated in 3 cohorts with doses ranging from 35 mg BID (1 patient) to 70 mg BID of BMS-354825. BMS-354825 is rapidly absorbed with peak concentrations achieved within 2 hours and a terminal phase half-life of about 5 hours. Consistent, rapid and sustained inhibition of LYN kinase, a member of the SRC family of tyrosine kinases, has been demonstrated. Of the 11 BP patients, 7 have had hematologic response: 3 complete hematologic response (CHR), 2 ‘no evidence of leukemia’ (NEL), and 2 ‘return to chronic phase’ (RTC). Three additional patients have had significant hematologic improvement despite being on treatment only a short period of time (10–23 days). One patient with extramedullary disease was stable. Cytogenetic data is available for 8 of the 11 BP patients. Four patients had major cytogenetic response, 2 patients had a minor cytogenetic response and 2 patients had no response. BCR-ABL mutation data is available for 2 patients: one patient did not have a mutation and one patient who had a non-sustained CHR was found to have a E355G mutation. Three of 6 AP patients have had hematologic response: 2 CHRs and 1 NEL. Two patients are too early to assess. One patient demonstrated resistance to BMS-354825 due to a T315I mutation in BCR-ABL found in 8 of 10 clones. This mutation confers resistance to BMS-354825 in preclinical studies. BCR-ABL mutation status is available for 3 additional AP patients: 2 patients had no mutations identified and 1 patient in CHR had M351T/A imatinib-resistant mutations. Of 3 patients for whom early cytogenetic data is available, 1 had a minor cytogenetic response (40% Ph+). To date BMS-354825 has been very well tolerated. Two patients in blast phase had evidence of mild tumor lysis syndrome. Dose escalation is continuing and phase II studies in chronic, accelerated and blast phase CML are currently being initiated. Further studies are required to establish LYN’s potential role in imatinib-resistant CML. The clinical data demonstrate that BMS-354825 can frequently override imatinib resistance in advanced CML, and provide compelling evidence supporting the safety and efficacy of BMS-354825 in imatinib-resistant accelerated and blast phase CML.

Even within comparatively recent years it was not uncommon to hear the question asked, “What is an Actuary”?; and, before dealing with our subject proper, it would therefore seem necessary, or very desirable at least, to trace to some extent the origin and development, so to speak, of the Actuary, and the rise and progress of the Actuarial Profession.

Many valuable papers have from time to time appeared among our own Transactions and in the Journal of the Institute of Actuaries bearing upon the Amounts invested by Life Offices in various securities, and the rates of interest yielded by such investments at different epochs. So far as I can discover, however, with the exception of a short, but very instructive, paper on Railway Debenture Stock by Mr. Coles in the 15th vol. of the Journal, nothing has been written in description of the origin and character of the various securities which are usually selected for the investment of Life Offices' and other Funds. Having discovered that many others besides myself have experienced difficulty in obtaining information on this subject, I have ventured to put down in an elementary way the results of my own research in regard to it.The President of the Institute of Actuaries, in his Inaugural Address, delivered recently, impressed upon his hearers the imperative necessity for the Actuary being now a practical financier also, and I am sure that every one of us knows sufficient about the difficulties that beset the investment of money at the present day to fully indorse his remarks. No one can, however, it seems to me, be a “practical financier” without having an intelligent knowledge of the character of various securities, and it is my desire to-night to try to help in some small measure towards the attainment of that knowledge.

The liability of the employer to compensate his employees, as well as other persons, for injuries sustained through his fault, may be traced from an early period in the world's history in the Common Law of various countries.For example, by the Jewish Law, said to have been promulgated about the year 1500 B.C., if a master were the means of causing the loss, either intentionally or unintentionally, of the eye or of the tooth of his slave, he was bound to let him go free for his eye or his tooth's sake. Again, according to the same law, if an employer allowed his ox to gore either his servant or a stranger, he was required to pay various compensations to the injured if he survived, or to his relatives in the event of the injury being followed by death.

Within quite recent years many schemes of life assurance without medical examination have been brought before the public by different offices, and have on the whole been very well received. No apology, therefore, seems necessary in submitting this paper to a meeting of the Faculty of Actuaries, more especially as, so far as we are aware, the matter has not previously been dealt with by any one.It would be a mistake to look upon our subject merely as one out of many innovations in assurance practice of late years. As every one knows, it belongs to the earliest times of life assurance, though it is only during the last few years that it has again been introduced into the sphere of practical business. While that is so, it must be admitted that the circumstances and conditions under which it existed formerly are very, or even altogether, different from those which have seemed to warrant or necessitate its resuscitation at the present day. To make this as clear as possible, it may be well, in the first place, to trace shortly, as far as it can be done, the origin and history of the medical examination of proposers for life assurance.

Reference has frequently been made to the fact that Life Assurance Companies are daily in the habit of undertaking liabilities which, in most cases, will not mature for a great number of years to come. As a rule, such deferment of the liability is welcomed by the offices as being in their view advantageous rather than otherwise. In most cases, however, it is not the office alone that undertakes liability. The policyholder on his part agrees to pay to the office a premium, it may be for a fixed number of years, should he live so long, or even during the remainder of his lifetime, no matter how long that may endure. It has been pointed out that, while the policyholder may at any time bring the engagement entered into between himself and the Assurance Office to an end without the latter's consent, the Company has usually no power to terminate the contract except with the concurrence of the policyholder.