Updates from the American Society of Hematology 2019 annual meeting: practice-changing studies in treatment-naïve chronic lymphocytic leukemia.

The 2019 annual meeting of the American Society of Hematology took place 7-10 December in Orlando, Florida. At the meeting, results from key studies in treatment-naïve chronic lymphocytic leukemia (cll) were presented. Of those studies, phase iii oral presentations focused on the efficacy and safety of therapy with inhibitors of Bruton tyrosine kinase (btk) and Bcl-2. One presentation reported updated results of the Eastern Cooperative Oncology Group 1912 trial comparing the efficacy and safety of ibrutinib-rituximab with that of fludarabine-cyclophosphamide-rituximab in patients less than 70 years of age with cll. A second presentation reported interim results of the elevate tn trial, which is investigating the efficacy and safety of acalabrutinib-obinutuzumab or acalabrutinib monotherapy compared with chlorambucil-obinutuzumab. A third presentation reported on the single-agent zanubrutinib arm of the sequoia trial in patients with del(17p). The final presentation constituted a data update from the cll14 trial, which is evaluating fixed-duration venetoclax-obinutuzumab compared with chlorambucil-obinutuzumab, including the association of minimal residual disease status with progression-free survival. Our meeting report describes the foregoing studies and presents interviews with investigators and commentaries by Canadian hematologists about potential effects on Canadian practice.

A Canadian perspective on bendamustine for the treatment of chronic lymphocytic leukemia and non-Hodgkin lymphoma.

Despite the success of standard treatments in chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL), patients are often unable to tolerate aggressive regimens, and they require effective alternatives. Bendamustine is a bifunctional alkylator with unique properties that significantly distinguish it from other agents in its class. In untreated CLL, bendamustine has demonstrated rates of response and progression-free survival (PFS) that are superior to those with chlorambucil, with an acceptable toxicity profile. In the relapsed setting, combination treatment with bendamustine-rituximab (BR) has demonstrated promising activity in high-risk patients such as those refractory to fludarabine or alkylating agents. In untreated patients with indolent NHL and mantle cell lymphoma, BR has demonstrated a PFS significantly longer than that achieved with R-CHOP (rituximab-cyclophosphamide-doxorubicin-vincristine-prednisone), with significantly reduced toxicity. In the relapsed setting, br has demonstrated rates of response and PFS superior to those with fludarabine-rituximab, with comparable toxicity. In the United States and Europe, bendamustine has been approved for the treatment of CLL and indolent NHL; its approval in Canada is pending and eagerly awaited. Once available, bendamustine will benefit many Canadian patients with NHL and CLL.

Early lymphocyte recovery following autologous peripheral stem cell transplantation is associated with better survival in younger patients with lymphoproliferative disorders.

Early absolute lymphocyte count (ALC) has become an important end point for engraftment in patients undergoing autologous peripheral stem cell transplantation (APSCT). In this retrospective study, we evaluate the prognostic significance of early recovery of ALC ( > or = 0.5 cells x 10(9)/l on or before day 15) following APSCT in predicting transplant outcome in 72 patients with lymphoproliferative disorders, including non-Hodgkin's lymphoma (n = 30), Hodgkin's lymphoma (n = 8) and multiple myeloma (n = 34). The median quantities of CD34+ stem cells and lymphocytes infused were 4.97 x 10(6)/kg (range 0.64-11.7) and 11.3 x 10(7)/kg (range 1.11-110) respectively. After a median follow-up of 18 months (range 2-68), 28 patients had experienced a relapse and 16 had died. Of the 72 patients, 27 (37%) demonstrated early recovery of ALC. Early recovery of ALC was strongly associated with long-term overall and disease-free survival in patients aged less than 50 years (P < 0.001). In both univariate and multivariate survival analyses, a shorter time from diagnosis to APSCT was associated with early recovery of ALC (P = 0.03). These findings indicate that early recovery of ALC may contribute to longer survival in younger patients with lymphoproliferative disorders. A shorter time from diagnosis to APSCT may favor recovery of ALC independent of the infused stem cell or lymphocyte doses.

Recommendations of the canadian consensus group on the management of chronic myeloid leukemia.

Chronic myelogenous leukemia (cml) is a disease characterized by the expression of Bcr/Abl, an oncogenic protein tyrosine kinase, and by evolution over time from a relatively benign chronic phase to a rapidly fatal cml blast crisis. Until recently, the standard of care included potentially curative therapy with allogeneic stem cell transplantation, available only to a minority (about 10%) of patients, or medical therapy with interferon-α with or without cytarabine, which helped to prolong the chronic phase of the disease in a minority of patients. The availability of imatinib mesylate, a selective inhibitor of Bcr/Abl approved by Health Canada in 2001, has profoundly altered the clinical and laboratory management of cml. This change in practice has been reviewed by the Canadian Consensus Group on the Management of Chronic Myelogenous Leukemia and has resulted in a new set of recommendations for the optimal care of cml patients.

Suppressed apoptosis of pre-B cells in bone marrow of pre-leukemic p190bcr/abl transgenic mice.

Mice transgenic for a p190bcr/abl construct develop pre-B cell leukemia/lymphoma, providing a model of Ph+ ALL. To investigate events in tumorigenesis, immunofluorescence labeling, flow cytometry and a short-term culture assay were used to quantitate precursor B cells and their apoptotic rates in bone marrow of p190bcr/abl transgenic mice over a wide age range. Malignancies appeared rapidly at 8-12 weeks of age, followed by slower tumor onset. At 8-12 weeks in normal mice, the apoptotic rate fell among pro-B cells but increased steeply among pre-B cells, while the total number of B lineage cells declined. In contrast, in p190bcr/abl transgenic mice over the same time period, while pro-B cells remained normal in apoptotic rate and number, apoptosis of pre-B cells was markedly inhibited and the number of B lymphocytes increased. At later ages (14-30 weeks), B cell precursors in control mice remained constant in apoptotic activity and number, while in the few surviving transgenic mice B cell populations were expanded. The results reveal characteristic changes in apoptotic activity among B cell precursors in bone marrow during early life, severely perturbed in preleukemic p190bcr/abl transgenic mice by a preferential suppression of pre-B cell apoptosis. p190bcr/abl may thus promote leukemogenesis by permitting aberrant cells generated during early B cell development to evade a normal quality checkpoint and negative selection.

Early events in leukemogenesis in P190Bcr-abl transgenic mice.

The activated tyrosine kinase, Bcr-abl, is implicated in a number of hematopoietic malignancies. The exact biological mechanism by which the kinases transforms cells is still not well delineated. Previous data has suggested that the inhibition of apoptosis and the deregulation of cell cycle progression as the result of P210Bcr-abl expression might contribute to leukemogenesis. In vitro systems in which Bcr-Abl is over-expressed have concluded that similar growth regulatory pathways are affected as a result of the expression of both P210 and P190Bcr-abl. Here, we utilized an in vitro P190Bcr-abl leukemia mouse model to dissect the early events that contribute to transformation by this isoform of Bcr-Abl. In this mouse model P190Bcr-abl is expressed as a low but physiologically relevant level in that all mice develop pre-B leukemia lymphomas. We show that cell cycle and apoptotic responses to DNA damage are intact in bone marrow and spleen cells of such animals. We also demonstrate a normal induction of p21WAF-1/CIP1 in both hematopoietic and non-hematopoietic tissue as a result of genotoxic stress. We suggest that P190Bcr-abl induced transformation is different than that of P210Bcr-abl.

Increase in mutant frequencies in mice expressing the BCR-ABL activated tyrosine kinase.

The acquisition of the Philadelphia (Ph) chromosome (or BCR-ABL translocation) represents a detrimental pathophysiological event in humans. The activated tyrosine kinases, which are produced by this translocation, are associated with fatal hematological malignancies. The initial molecular dissection of BCR-ABL has linked the expression of this constitutively activated kinase with enhanced genomic instability. We directly evaluated the consequence of BCR-ABL expression on genomic instability using the Big Blue in vivo mutagenesis mouse system. We report that the expression of BCR-ABL in both spleens and kidneys confers a mutator phenotype represented by a statistically significant elevation in mutant frequencies.

P210 Bcr-Abl interacts with the interleukin-3 beta c subunit and constitutively activates Jak2.

Chronic myelogenous leukemia is a neoplasm of pluripotent hematopoietic cells. Cytokines such as interleukin-3 and granulocyte-macrophage colony-stimulating factor regulate the growth and differentiation of hematopoietic precursors. These cytokines activate two distinct signals to the nucleus. One signal is through the Ras pathway, and the second involves activation of Jak2. We demonstrated that Bcr-Abl co-immunoprecipitates with and constitutively phosphorylates the common beta c chain of the interleukin-3 (IL-3) and granulocyte-macrophage-macrophage colony-stimulating factor (GM-CSF) receptors. Our data show that formation of this complex leads to the constitutive activation of Jak2. Previously, it has been demonstrated that Bcr-Abl interacts with Grb2 and Shc, which in turn activates the Ras pathway. Thus, Bcr-Abl can activate signalling through both pathways in a factor-independent fashion.

Effect of chronic renal failure on the expression of erythropoietin message in a murine model.

The anemia of chronic renal failure (CRF) is largely due to decreased production of erythropoietin (EPO) by the kidney. A small amount of EPO also originates from extra-renal sources, and this would be expected to assume a more important role in maintaining erythropoiesis when renal production is impaired. In this study, we examined the production of EPO mRNA by RT-PCR in kidney, liver, and bone marrow tissues isolated from normal mice, mice rendered acutely anemic by phlebotomy, and from mice with surgically induced CRF. The induction of acute anemia results in an expected increase in the expression of EPO mRNA in renal and hepatic tissue. In contrast, while the expression of EPO mRNA was expectedly reduced in the kidney from CRF mice, it was completely absent in the liver of these same animals. EPO mRNA expression was also absent in the bone marrow in both states of acute anemia and CRF. These results show that CRF can directly or indirectly can suppress the extrarenal production of EPO by the liver and that this effect may further aggravate the anemia of CRF.

P210 Bcr-Abl interacts with the interleukin 3 receptor beta(c) subunit and constitutively induces its tyrosine phosphorylation.

Chronic myelogenous leukemia is a neoplasm of pluripotent hematopoietic cells. The P210 Bcr-Abl oncoprotein is a deregulated cytoplasmic tyrosine kinase that has been shown to cause chronic myelogenous leukemia-like neoplasms in mice. Cytokines such as interleukin 3 and granulocyte/macrophage-colony-stimulating factor regulate the growth and differentiation of hematopoietic precursors. These cytokines activate two distinct signals to the nucleus. One signal is through the Ras pathway, and the second involves activation of Jak2. We demonstrated that Bcr-Abl co-immunoprecipitates with, and constitutively phosphorylates, the common beta(c) subunit of the interleukin 3 and granulocyte/macrophage-colony-stimulating factor receptors. Our data show that formation of this complex leads to the constitutive tyrosine phosphorylation of Jak2. It has been demonstrated that Bcr-Abl interacts with Grb2 and Shc, which in turn activates the Ras pathway. Our new findings raise the possibility that Bcr-Abl activates signaling through both pathways in a factor-independent fashion.

Abl tyrosine protein kinase.

Mammalian c-Abl belongs to an evolutionary conserved family of non-receptor tyrosine kinases. It is distributed both in the cytoplasm in association with F-actin, and in the nucleus where it binds chromatin. The normal function of c-Abl is poorly understood. Nevertheless, there has been rapid progress in the characterization of the structural features, signal transduction pathways, substrates and ligands involved in the action of c-Abl and Abl-derived oncogenes. These developments suggest that several mechanisms co-operate to allow regulation of normal cell growth by c-Abl and induction of leukemias by Bcr-abl.

Expression of bcr-abl abrogates factor-dependent growth of human hematopoietic M07E cells by an autocrine mechanism.

The introduction of a retrovirus vector expressing p210bcr-abl (P210) into the human factor-dependent cell line M07E resulted in the rapid outgrowth of factor-independent cells. Early after infection, four factor-independent clones were isolated and analyzed in greater detail along with mass populations obtained from separate infections. High levels of P210 tyrosine kinase activity were measured in the factor-independent cells. The mass populations and three of the four clones remained responsive to exogenous growth factors. Concentrated conditioned media isolated from the factor-independent populations and from all clones contained biologically active granulocyte-macrophage colony-stimulating factor (GM-CSF); interleukin-3 (IL-3) was detected at low levels in the mass population and in two of the clones. Neutralizing antibodies to IL-3, GM-CSF, and mast cell growth factor inhibited proliferation of the factor responsive clones by 60% to 90%. These results indicate that the growth autonomy of the P210-expressing M07E cells was acquired via an autocrine mechanism. In addition to factor-independent growth, P210-expressing M07E cells readily acquired a more mature megakaryocytic phenotype compared with control M07E cells. These data provide experimental evidence that expression of P210 tyrosine kinase in human hematopoietic cells induced growth factor secretion resulting in a pleiotropic effect on growth factor dependence and differentiation.

bcr/abl expression in 32D cl3(G) cells inhibits apoptosis induced by protein tyrosine kinase inhibitors.

Eight protein tyrosine kinase inhibitors with in vitro epidermal growth factor receptor kinase 50% inhibitory concentration values ranging from 0.043 to 22 microM were studied for their ability to inhibit the growth of the murine interleukin-3 (IL-3) dependent myeloid 32D cl3(G) cell line and, a subclone (LG7) transformed to IL-3 independent growth by retroviral transduction and expression of the chronic myelogenous leukemia-associated protein tyrosine kinase p210bcr/abl. Cell proliferation 50% inhibitory concentration values ranged from 4 to 250 microM, and one compound was not inhibitory at 500 microM. The dose-cell proliferation curves were remarkably similar for parental 32D cl3(G) cells + IL-3 and LG7 +/- IL-3, and reversion of LG7 cells to IL-3 dependence was not observed, suggesting that none of the compounds tested could selectively inhibit p210bcr/abl. However, 6 compounds induced the appearance of a 200-base pair nucleosomal DNA ladder characteristic of apoptosis at 24 h in parental 32D cl3(G) cells + IL-3, which mimicked the effects of IL-3 withdrawal alone, but not in similarly growth arrested LG7 cells that eventually developed a necrotic pattern of DNA fragmentation. These studies suggest that the expression of p210bcr/abl can suppress apoptotic signal transduction and that this may contribute to the development of the myeloid hyperplasia that occurs in chronic phase chronic myelogenous leukemia.

Expression of p210 bcr/abl increases hematopoietic progenitor cell radiosensitivity.

PURPOSE: The cytogenetic finding of the Ph1+ chromosome and its molecular biologic marker bcr/abl gene rearrangement in cells from patients with chronic myeloid leukemia are associated with a proliferative advantage of the Ph1+ clone in vivo. Although the transition to the acute terminal phase or blastic crisis is often associated with additional cytogenetic abnormalities, the molecular events which correlate the initial cytogenetic lesion with the terminal phase are poorly understood. Defective cellular DNA repair capacity is often associated with chromosomal instability, increased mutation frequency, and biologic alterations. METHODS AND MATERIALS: We, therefore, tested whether the protein product of the bcr/abl translocation (p210) could alter DNA repair after gamma-irradiation of murine cell lines expressing the bcr/abl cDNA. RESULTS: The 32D cl 3 parent, 32D cl 3 pYN (containing the control vector plasmid) and each of two sources of 32D cl 3 cells expressing p210 bcr/abl cDNA (32D-PC1 cell line and 32D-LG7 subclone) showed a D0 of 1.62, 1.57, 1.16, and 1.27 Gy, respectively. Thus, expression of the p210 bcr/abl product induced a significant (p < 0.05) increase in radiosensitivity at the clinically relevant radiation therapy dose-rate (1.16 Gy/min). The increased radiosensitivity of p210 bcr/abl expressing cells persisted if cells were held before plating in a density-inhibited state for 8 hr after gamma-irradiation, indicating little effect on the repair of potentially lethal gamma-irradiation damage. The IL-3 dependent parent 32D cl 3 cells demonstrated programmed cell death in the absence of growth factor or following gamma-irradiation to 200 cGy. Expression of bcr/abl cDNA in the 32D-PC1 and 32D-LG7 sub clones abrogated IL-3 requirement of these cell lines and inhibited gamma-irradiation induced programmed cell death. CONCLUSION: These data suggest a role for bcr/abl p210 in amplifying gamma-irradiation DNA damage or broadly inhibiting DNA repair, conditions that may stimulate further cytogenetic alterations in hematopoietic cells.

Clonal evolution in a myeloid cell line transformed to interleukin-3 independent growth by retroviral transduction and expression of p210bcr/abl.

Current evidence suggests that the expression of the tyrosine kinase p210bcr/abl in chronic myelogenous leukemia (CML) may directly induce the initial phase of granulocytic hyperplasia. However, the dysregulation of additional genes appears to be required for transition to the acute leukemic phase, as inferred by the appearance of recurrent secondary cytogenetic abnormalities in the majority of patients. To determine whether the expression of p210bcr/abl alone is responsible for this genetic instability, we introduced and expressed the bcr/abl gene from a retroviral vector in a clone of the interleukin-3 (IL-3) dependent myeloblastic 32D C13(G) cell line. Clonal and polyclonal cells transformed to IL-3 independent growth were observed for a period extending up to 6 months for changes in the expression of p210bcr/abl, cell proliferation, inhibition by prostaglandin E1 (PGE1), forskolin, and cyclic adenosine monophosphate (cAMP) analogues, regulation of the cell cycle, and karyotype. Whereas the properties of control vector infected 32D C13(G)' cells remained stable over time, cells expressing p210bcr/abl were phenotypically unstable. In cells expressing p210bcr/abl, we observed selective modulation of p210bcr/abl mRNA and protein expression, evolution from partial to full abrogation of IL-3 dependence, reduced serum requirements, increased cell proliferation, decreased inhibition by PGE1 and cAMP analogues, and the appearance of new structural and numerical chromosomal abnormalities with successive cell passages. These results indicate that expression of p210bcr/abl can directly predispose 32D C13(G)' cells to genetic instability, promotes the emergence of clones with an increased proliferative advantage, and may represent an in vitro model suitable for the study of mechanisms underlying progression to the acute leukemic phase in CML.

Enhanced growth of a human keratinocyte cell line induced by antisense RNA for parathyroid hormone-related peptide.

We have used antisense RNA technology to inhibit endogenous parathyroid hormone-related peptide (PTHRP) production in the established human keratinocyte cell line, HPK1A, in order to assess the role of PTHRP as a potential modulator of cell growth. Rat PTHRP cDNA was cloned into the replication defective retroviral vector pYN in an antisense orientation and a stable cell line (HPK1A-AS) was generated after infection by amphotropic virus and selection by the neomycin derivative, G418. Expression of the transfected antisense sequence was confirmed with an RNA sense probe for PTHRP. The effect of the retrovirally mediated gene transfer on the endogenous PTHRP transcript was examined with an RNA antisense probe which demonstrated an absence of the endogenous transcript in HPK1A-AS cells. A 1.6-kilobase transcript was, however, present in equivalent quantities in both uninfected HPK1A and pYN-infected (HPK1A-pYN) cells. Immunocytochemistry and assessment of PTHRP secretion into the medium using an NH2-terminal radioimmunoassay and a UMR 106 adenylate cyclase bioassay confirmed the absence of PTHRP in HPK1A-AS cells. Examination of the inhibition of PTHRP production on cell growth demonstrated a reduction in doubling time and an increase in [3H]thymidine incorporation. Cell cycle analysis showed an increase in the proportion of the cell population in the S phase (relative to G0/G1) in HPK1A-AS cells compared to HPK1A or HPK1A-pYN cells. These data, therefore, indicate that endogenous PTHRP acts as an effective inhibitor of cell growth in this keratinocyte model and that this action occurs, at least in part, by diminishing entry into the S phase of the cell cycle. Furthermore, the antisense RNA method is a potent one to evaluate the cellular actions of PTHRP.

Clonal succession and deletion of bcr/abl sequences in chronic myelogenous leukemia with recurrent lymphoid blast crisis.

The development of cancer is generally believed to occur by a multistep process in which critical genetic defects accumulate in a clone of cells, confer a growth advantage, and result in the emergence of more malignant subclones. This paper describes the clonal origin of cells in a patient with Philadelphia-chromosome negative, M-bcr rearrangement-positive chronic myelogenous leukemia, observed in two episodes of lymphoid blast crisis (BC), the intervening chronic phases (CP), and following allogeneic bone marrow transplantation. Serial analysis of immunoglobulin heavy and kappa light chain (IgJH, IgCK), beta-T-cell receptor (beta-TcR) and bcr major breakpoint cluster region (M-bcr) gene rearrangements was performed. Clonal IgJH rearrangements present in cells of the first lymphoblastic crisis (BC1) were altered during the chronic phase post-treatment (CP1), and were again altered in recurrent blast crisis (BC2). In addition, the M-bcr gene rearrangement present in BC1 and CP1 was absent from cells in BC2. These observations suggest that the course of clinical neoplastic disorders may not always be characterized simply by a hierarchical process of clonal evolution, but may also involve clonal succession of malignant cells. Moreover, the deletion of M-bcr in recurrent BC suggests that bcr/abl may not be essential for the maintenance of cell growth in established BC.

Expression of the chronic myelogenous leukemia-associated p210bcr/abl oncoprotein in a murine IL-3 dependent myeloid cell line.

We have studied the effect of a replication-defective murine retroviral vector expressing the chronic myelogenous leukemia associated oncoprotein p210bcr/abl in murine IL-3 dependent myeloid 32D C13(G) cells. This cell line can be induced to differentiate along either the granulocytic or monocytic lineages thus permitting an independent assessment of the effect of p210bcr/abl on growth and differentiation. Cells expressing p210bcr/abl displayed a complete non-autocrine abrogation of IL-3 dependence and an enhanced response to an activity in FBS which is not IGF-I or IGF-II. During the first few generations following infection with the bcr/abl vector, cells became larger with an increased fraction of cells in G2/M and monocyte/macrophage markers were expressed. Four cytoplasmic proteins phosphorylated in response to IL-3 in the parental cell line with apparent molecular weights of 98, 70, 62, and 52 Kd were amongst those constitutively phosphorylated in p210bcr/abl expressing cells. These results suggest that the functional substitution of IL-3 by p210bcr/abl is due to constitutive activation of proteins involved in IL-3 signal transduction. Alterations of cell differentiation, cell cycle and growth which cannot be attributed to IL-3 like effects indicate that p210bcr/abl has pleiotropic effects involving several other pathways of cellular regulation.