Eibhlin Conneally

Optimization of retroviral-mediated gene transfer to human NOD/SCID mouse repopulating cord blood cells through a systematic analysis of protocol variables

Retroviral transduction of human hematopoietic stem cells is still limited by lack of information about conditions that will maximize stem cell self-renewal divisions in vitro. To address this, we first compared the kinetics of entry into division of single human CD34+CD38- cord blood (CB) cells exposed in vitro to three different flt3-ligand (FL)-containing cytokine combinations. Of the three combinations tested, FL + hyperinterleukin 6 (HIL-6) yielded the least clones and these developed at a slow rate. With either FL + Steel factor (SF) + HIL-6 + thrombopoietin (TPO) or FL + SF + interleukin 3 (IL-3) + IL-6 + granulocyte-colony-stimulating factor (G-CSF), >90% of the cells that formed clones within 6 days undertook their first division within 4 days, although not until after 24 hours. These latter two, more stimulatory, cytokine combinations then were used to assess the effect of duration of cytokine exposure on the efficiency of transducing primitive CB cells with a gibbon ape leukemia virus-pseudotyped murine retroviral vector containing the enhanced green fluorescent protein (GFP) cDNA and the neomycin resistance gene. Fresh lin- CB cells exposed once to medium containing this virus plus cytokines on fibronectin-coated dishes yielded 23% GFP+ CD34+ cells and 52-57% G418-resistant CFC when assessed after 2 days. Prestimulation of the target cells (before exposing them to virus) with either the four or five cytokine combination increased their susceptibility. In both cases, the effect of prestimulation assessed using the same infection protocol was maximal with 2 days of prestimulation and resulted in 47-54% GFP+ CD34+ cells and 67-69% G418-resistant CFC. Repeated daily addition of new virus (up to three times), with assessment of the cells 2 days after the last addition of fresh virus, gave only a marginal improvement in the proportion of transduced CD34+ cells and CFC, but greatly increased the proportion of transduced LTC-IC (from 40% to >99%). Transplantation of lin- CB cells transduced using this latter 6-day protocol into NOD/SCID mice yielded readily detectable GFP+ cells in 10 of 11 mice that were engrafted with human cells. The proportion of the regenerated human cells that were GFP+ ranged from 0.2-72% in individual mice and included both human lymphoid and myeloid cells in all cases. High-level reconstitution with transduced human cells was confirmed by Southern blot analysis. These findings demonstrate that transplantable hematopoietic stem cells in human CB can be reproducibly transduced at high efficiency using a 6-day period of culture in a retrovirus-containing medium with either FL + SF + HIL-6 + TPO or FL + SF + IL-3 + IL-6 + G-CSF in which virus is added on the third, fourth, and fifth day.

The Vascular Targeting Agent Combretastatin-A4 and a Novel cis-Restricted β-Lactam Analogue, CA-432, Induce Apoptosis in Human Chronic Myeloid Leukemia Cells and Ex Vivo Patient Samples Including Those Displaying Multidrug Resistance

Combretastatin-A4 (CA-4) is a natural derivative of the African willow tree Combretum caffrum. CA-4 is one of the most potent antimitotic components of natural origin, but it is, however, intrinsically unstable. A novel series of CA-4 analogs incorporating a 3,4-diaryl-2-azetidinone (β-lactam) ring were designed and synthesized with the objective to prevent cis -trans isomerization and improve the intrinsic stability without altering the biological activity of CA-4. Evaluation of selected β-lactam CA-4 analogs demonstrated potent antitubulin, antiproliferative, and antimitotic effects in human leukemia cells. A lead β-lactam analog, CA-432, displayed comparable antiproliferative activities with CA-4. CA-432 induced rapid apoptosis in HL-60 acute myeloid leukemia cells, which was accompanied by depolymerization of the microtubular network, poly(ADP-ribose) polymerase cleavage, caspase-3 activation, and Bcl-2 cleavage. A prolonged G2M cell cycle arrest accompanied by a sustained phosphorylation of mitotic spindle checkpoint protein, BubR1, and the antiapoptotic proteins Bcl-2 and Bcl-xL preceded apoptotic events in K562 chronic myeloid leukemia (CML) cells. Molecular docking studies in conjunction with comprehensive cell line data rule out CA-4 and β-lactam derivatives as P-glycoprotein substrates. Furthermore, both CA-4 and CA-432 induced significantly more apoptosis compared with imatinib mesylate in ex vivo samples from patients with CML, including those positive for the T315I mutation displaying resistance to imatinib mesylate and dasatinib. In summary, synthetic intrinsically stable analogs of CA-4 that display significant clinical potential as antileukemic agents have been designed and synthesized.

Allogeneic Hematopoietic Stem Cell Transplantation for a BCR-FGFR1 Myeloproliferative Neoplasm Presenting as Acute Lymphoblastic Leukemia

Hematopoietic myeloproliferative neoplasms (MPNS) with rearrangements of the receptor tyrosine kinase FGFR1 gene, located on chromosome 8p11, are uncommon and associated with diverse presentations such as atypical chronic myeloid leukemia, acute myeloid leukemia, or an acute T- or B-lymphoblastic leukemia, reflecting the hematopoietic stem cell origin of the disease. A review of MPN patients with the t(8;22) translocation that results in a chimeric BCR-FGFR1 fusion gene reveals that this disease either presents or rapidly transforms into an acute leukemia that is generally unresponsive to currently available chemotherapeutic regimens including tyrosine kinase inhibitors (TKIS). The first case of a rare BCR-FGFR1 MPN presenting in a B-acute lymphoblastic phase who underwent allogeneic hematopoietic stem cell transplantation (HSCT) with a subsequent sustained complete molecular remission is described. Allogeneic HSCT is currently the only available therapy capable of achieving long-term remission in BCR-FGFR1 MPN patients.

Clonal karyotypic abnormalities in Philadelphia negative cells of CML patients treated with imatinib: is it under-reported and does it have any clinical significance?

Froom, P., Elmalah, I., Braester, A., Aghai, E. & Quitt, M. (2002) Clodronate in myelofibrosis: a case report. American Journal of Medical Sciences, 332, 115–116. Sivera, P., Cesano, L., Guerrasio, A., Camaschella, C. & Mazza, U. (1994) Clinical and haematological improvement induced by etidronate in a patient with idiopathic myelofibrosis and osteosclerosis. British Journal of Haematology, 86, 397–398.

Evaluation of a JAK2 V617F quantitative PCR to monitor residual disease post-allogeneic hematopoietic stem cell transplantation for myeloproliferative neoplasms.

*Corresponding author: Karl Haslam, Cancer Molecular Diagnostics, Central Pathology Laboratory, St. James’s Hospital, Dublin 8, Ireland, Phone: +353 1 4103575, Fax: +353 1 4103513, E-mail: khaslam@stjames.ie Karen M. Molloy and Stephen E. Langabeer: Cancer Molecular Diagnostics, Central Pathology Laboratory, St. James’s Hospital, Dublin, Ireland Eibhlin Conneally: Department of Haematology, St. James’s Hospital, Dublin, Ireland

Chronic myeloid leukaemia presenting post-radiotherapy for prostate cancer: further evidence for an immunosurveillance effect.

T-lymphocytes are known to have a fundamental role in the immunological control of chronic myeloid leukaemia (CML). Experience from stem cell transplantation has shown that recipients of T cell-depleted grafts have a higher incidence of relapse, and in those patients who relapse post-transplant, remissions can be induced by donor lymphocyte infusions. Sizeable T cell clones including specific cytotoxic T cells (CTL) that recognize leukaemia-associated antigens are present in a significant number of CML patients at diagnosis (Kreutzman et al, 2010), which may afford an opportunity for targeted immunotherapy (Riley et al, 2009). According to the immunosurveillance hypothesis, expansion of an occult malignant clone requires escape from immune recognition whereby effector cells are either deleted or rendered anergic: a function that is predominantly performed by the natural killer cells of the innate immune system (Waldhauer & Steinle, 2008). A number of cancer treatments, such as chemotherapy, radiotherapy, surgery and hormonal regulation, can all result in suppression of this innate cytotoxicity. Evidence for an immunosurveillance effect that prohibits development of CML derives from the observations of low levels of BCR-ABL1 transcripts present in normal, healthy individuals (Biernaux et al, 1995; Bose et al, 1998) and from an observed increase in the frequency of CML in longterm immunosuppressed recipients of organ transplants (le Coutre et al, 2010). A case is described in which CML manifested rapidly after radical radiotherapy for prostate cancer and which provides further evidence for an immunosurveillance mechanism, that when deregulated, resulted in clonal expansion and subsequent development of overt CML. A 62-year-old male was referred to the haematology service for investigation of a modest eosinophilia (maximally 0 9 9 10/l) and progressive basophilia (0 2–0 7 9 10/l) with the remainder of his complete blood count (CBC) normal. Retrospective CBCs for the previous 3 years were available (Fig 1). His only notable symptom was mild pruritus of the lower limbs. He was diagnosed with prostate cancer 3 years previously for which he underwent a radical prostactectomy. His prostate-specific antigen (PSA) had remained stable for 2 years post-surgery but had begun to rise; hence he received radical radiotherapy (66 6 Gy) to the prostate bed with a subsequent return to normal of the PSA. Approximately 3 months post-radiotherapy he was noted to have a leucocytosis (22 3 9 10/l) including eosinophilia

Monitoring Residual Disease in the Ph-Negative Myeloproliferative Neoplasms Post-Allogeneic Stem Cell Transplantation: More Mutations and More Methodologies

The Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic diseases characterized by bone marrow proliferation of one or more of the myeloid cell lineages with no marked alterations in cellular maturation. MPN classically comprise the clinically and pathologically related polycythemia vera (PV), essential thromobocythemia (ET), and primary myelofibrosis (PMF). Identification of the JAK2 V617F mutation has revolutionized the molecular diagnosis of MPN as this mutation is present in >95% of patients with PV and in 50% of patients with ET and PMF. In PV and ET, the potential exists for the disease to transform to a myelofibrotic stage and, together with PMF, transformation to acute myeloid leukemia (AML). The introduction of small molecule inhibitors that abrogate both normal and mutant JAK protein intracellular signaling in PMF has undoubtedly been a major advance in the treatment of these types of malignancies (1). Nevertheless, the only potentially curative option for these diseases, particularly PMF, is allogeneic stem cell transplantation (ASCT). Though ASCT was previously considered only for those patients with advanced or transformed disease, improvements in candidate patient selection and stratification, timing of transplantation, and conditioning regimens have significantly reduced the transplant related morbidity and increased the overall survival for MPN patients undergoing this procedure (2). However, as relapse is a major cause of treatment failure post-ASCT with salvage options limited and subsequent outcome relatively poor, identification of those patients at high-risk of relapse would be highly desirable, potentially enabling therapeutic intervention before overt relapse. Conventionally, donor chimerism status is used to assess engraftment post-ASCT for hematological malignancies. Comparison of donor and recipient profiles can be achieved by either short tandem repeat (STR) analysis or quantitative polymerase chain reaction (qPCR) possessing sensitivities of 1–2%, with post-ASCT surveillance performed at one to three-monthly intervals. Post-ASCT monitoring utilizing additional patient-specific markers is most likely to provide a more beneficial, personalized profile with this approach already applied to many MPN patients undergoing ASCT. Early studies demonstrated the achievement of a molecular remission in JAK2 V617F-positive MPN post-ASCT using qualitative PCR (3). Development of more sensitive, JAK2 V617F-specific qPCR methodologies capable of detecting one mutant allele in 104 wild type copies has subsequently been shown to provide information on the rate of disease eradication and the identification of patients, at defined time points post-ASCT, at an increased risk of relapse (4–6). These sensitive JAK2 V617F qPCR assays have also been shown to be of value in triggering adoptive immunotherapies such as donor lymphocyte infusions that are able to elicit a graft-versus-tumor effect both preemptively and for salvage, post-relapse (7, 8). Even so, such qPCR methodologies require optimization across platforms and rigorous attention to reliability and sensitivity to ensure continued clinical utility (9). Mutations of MPL that encodes the receptor for thrombopoietin are also recurrent in ET and PMF but at a much lower frequency than the JAK2 V617F. In those reported MPL-mutation positive MPN who have undergone ASCT, rapid clearance of the MPL W515L mutation correlated well with peripheral blood counts and donor chimerism status (10). More recently, whole exome sequencing has identified insertion and/or deletion mutation in CALR, a gene that encodes the endoplasmic reticulum-associated, calcium binding protein calreticulin. These mutations, which occur exclusively in CALR exon nine, appear not to be found in PV, and are present in up to 80% of ET and PMF patients who are JAK2 V617F-and MPL-negative (11, 12). As CALR mutations are likely initiating events in MPN pathogenesis, the possibility arises to assess these mutations as markers of residual disease in MPN patients post-ASCT. An initial assessment of CALR mutant allele burden, using semi-quantitative PCR fragment analysis, has shown that eradication and persistence of CALR mutations mirror donor chimerism status providing initial validation for future prospective serial monitoring of CALR mutations post-ASCT (13). Apart from the common JAK2 V617F, MPL, and CALR mutations, several other recurrent mutations are observed in MPN but are also present in the myelodysplastic syndromes (MDS), MDS/MPN syndromes, and AML limiting their diagnostic utility but affording the potential for their use as markers of residual disease. These include mutations of TET2, ASXLI, CBL, NRAS, and EZH2. Rarely, mutations within some of these genes have been demonstrated in normal individuals (14) advocating caution when employed as markers of residual disease. Next generation sequencing (NGS) technologies now afford the opportunity to identify pathogenic mutations in multiple amplicons from these and many other genes simultaneously. A recent proof of principle study has utilized NGS to identify molecular mutations in MDS/MPN patients pre-ASCT with detection of the corresponding mutation in the post-ASCT period predictive of relapse (15). Whereas qPCR techniques provide relative quantitation of target amplicons, the emerging technology of digital PCR (dPCR) allows the absolute quantitation of mutant alleles with very small fold changes quantifiable. This approach has been applied to detect and monitor extremely low levels of BCR-ABL1 transcripts in chronic myeloid leukemia and acute lymphoblastic leukemia patients who have achieved deep molecular responses with tyrosine kinase inhibitor therapy (16, 17). Whether NGS technologies will be of value in routine practice in the post-ASCT setting remains to be proven. Currently, limits of detection require improvement in order to approach those achieved by qPCR necessary for clinical application (Figure ​(Figure1).1). Furthermore, issues such as intra- and inter-laboratory will require addressing for both NGS and dPCR; preliminary studies with NGS have demonstrated technical feasibility and concordance in the diagnostic setting (18). These emerging methodologies might soon be applied to identify and monitor mutational events enabling a distinct, personalized profile in those MPN patients undergoing ASCT. The goal would be the prompt recognition of those patients with an increased risk of relapse in which early therapeutic intervention is warranted, ultimately leading to gains in long-term disease-free and overall survival. Figure 1 Summary of methodologies for monitoring MPN-specific mutations post-allogeneic stem cell transplantation. ASCT, allogeneic stem cell transplantation; MPN, myeloproliferative neoplasm; qPCR, quantitative polymerase chain reaction; NGS, next generation ...

BCR-ABL1 kinase domain mutation analysis in an Irish cohort of chronic myeloid leukemia patients.

While tyrosine kinase inhibitor (TKI) therapy is the mainstay of modern management of chronic myeloid leukemia (CML), a significant proportion of CML patients may be refractory or lose their initial response to TKI therapy through a number of cellular and molecular mechanisms of which acquired mutations in the BCR-ABL1 kinase domain (KD) are the most common. BCR-ABL1 KD mutations were prospectively identified in order to inform clinical decisions on subsequent therapy. Direct sequencing of the BCR-ABL1 KD was performed in 85 CML patients that were either TKI refractory or displayed increasing BCR-ABL1 transcript levels by serial monitoring after an initial molecular response. Twenty-three BCR-ABL1 KD mutations were detected in 21 CML patients and were detected across the KD. Mutations were associated with specific TKI resistance, indicating change and enabling rational selection of subsequent therapy. Serial molecular monitoring of BCR-ABL1 transcripts in CML patients allows appropriate selection of CML patients for BCR-ABL1 KD mutation analysis associated with acquired TKI resistance. Identification of these KD mutations is essential in order to direct alternative treatment strategies in such CML patients.

Myelodysplastic Syndrome/Acute Myeloid Leukemia Arising in Idiopathic Erythrocytosis

The term “idiopathic erythrocytosis (IE)” is applied to those cases where a causal clinical or pathological event cannot be elucidated and likely reflects a spectrum of underlying medical and molecular abnormalities. The clinical course of a patient with IE is described manifesting as a persistent erythrocytosis with a low serum erythropoietin level, mild eosinophilia, and with evidence of a thrombotic event. The patient subsequently developed a myelodysplasic syndrome (MDS) and acute myeloid leukemia (AML), an event not observed in erythrocytosis patients other than those with polycythemia vera (PV). Application of a next-generation sequencing (NGS) approach targeted for myeloid malignancies confirmed wild-type JAK2 exons 12–15 and identified a common SH2B3 W262R single-nucleotide polymorphism associated with the development of hematological features of myeloproliferative neoplasms (MPNs). Further NGS analysis detected a CBL L380P mutated clone expanding in parallel with the development of MDS and subsequent AML. Despite the absence of JAK2, MPL exon 10, or CALR exon 9 mutations, a similarity with the disease course of PV/MPN was evident. A clonal link between the erythrocytosis and AML could be neither confirmed nor excluded. Future molecular identification of the mechanisms underlying IE is likely to provide a more refined therapeutic approach.

Characterization of a novel variant BCR-ABL1 fusion transcript in a patient with chronic myeloid leukemia: Implications for molecular monitoring.

Molecular monitoring of BCR-ABL1 transcript levels using quantitative polymerase chain reaction is an essential part of the modern management of chronic myeloid leukemia patients treated with tyrosine kinase inhibitors. Establishing the diagnostic BCR-ABL1 fusion transcript is necessary in order to select appropriate primers and probes for such monitoring. A case is described in which quantitative polymerase chain reaction failed to detect the presence of BCR-ABL1 fusion transcript in a Philadelphia chromosome-positive chronic myeloid leukemia patient. Further investigation demonstrated a novel in-frame BCR-ABL1 fusion transcript with a breakpoint in BCR exon 13 and insertion of a sequence of ABL1 intron 1, therefore enabling subsequent molecular monitoring. This case highlights the requirement for characterization of the BCR-ABL1 transcript type at chronic myeloid leukemia diagnosis. Issues concerning standardized methodological approaches and interpretation of transcript levels in such rare cases are discussed.