ALBERT

Description: Introduction: Interphase FISH on CD138-selected bone marrow cells enables genetic risk stratification in newly diagnosed multiple myeloma (MM), however as MM remains incurable, most centres still treat newly diagnosed MM uniformly, utilising the most active regimens available. At relapse an increasing choice of regimens, coupled with co-morbidities and treatment-emergent toxicities, means no uniform approach is possible. Instead, therapy is tailored to disease and patient related risk factors. In this setting, FISH testing may be particularly useful if not done at diagnosis and to identify progression events that may alter prognosis. Aim: To evaluate the outcome of FISH analysis in consecutive patients with relapsed MM undertaken at our centre: success rate, frequency of abnormalities, incidence of progression events and correlation of FISH abnormalities with treatment outcomes. Methods: FISH analysis was performed on 192 samples from 154 relapsed patients (2012-13). Plasma cells were selected using magnetic CD138 MicroBeads and interphase FISH carried out using probes as recommended by the EMN (Ross et al, 2012). If patients had no prior results, a full FISH MM panel was performed, using probes for t(4;14), t(14;16), t(11;14), deletion 17p (17p-), Chr 1 abnormalities (1p-/1q+) and deletion 13q (13q-). If patients had been previously tested for an IgH translocation (Tx), a progression event panel was used: 1p-/1q+, 17p- and 13q-. Patients underwent FISH testing prior to starting the next line of therapy. Results: 79% of samples were successfully analysed, with analysis limited in 16% and failed in 5%. Common reasons for failure were poor quality/aged slides, insufficient material and poor hybridisation. 17% of patients had no cytogenetic abnormality. The most common abnormality was 13q- (43.1%), followed by 1q+ (41.4%), t(11;14) (18.3%), t(4;14) (12.4%), 17p- (12.0%) 1p- (8.9%), and t(14;16) (5.6%) Progression events were more common in t(14;16) and t(4;14) groups. All patients with t(14;16) and 82% with t(4;14) had an additional genetic lesion. Only 21% of patients with t(11;14) and 54% with no IgH Tx had an additional event. 80 patients (51.3%) had prior FISH results and 13 (16.3%) had developed a new abnormality on the later test. In 9 cases the progression event was 17p-, in 2 it was 1q+ and 2 cases developed 17p- and 1q+. The patients developing 1q+ were previously standard risk, so repeat testing altered risk group. Acquisition of 17p- indicates especially poor outcome, thus in all 13 cases repeat FISH analysis altered risk. Among patients with progression events none harboured t(11;14), 8 (64%) had no IgH Tx, 3 had t(14;16) and 2 had t(4;14). FISH results were correlated with clinical outcome. Patients were stratified as having high risk genetics [t(4;14), t(14,16), 17p- in ≥50% cells, 1p-/1q+] or standard risk [t(11;14), normal cytogenetics]. 63 (41%) patients were high risk, 83 (54%) standard risk, with no information available for 8 (5%). Both groups had received a median of 2 prior lines of therapy. Response rates (≥PR) to the next line of therapy were similar (60.4% standard risk vs 56.0% high risk). PFS from time of FISH was significantly longer in the standard risk group (9.8 months vs 5.9, p

Description: Chimeric antigen receptor (CAR)19 T-cells exhibit powerful anti-leukemic effects in patients with B cell malignancies. However, the complexity of production of patient bespoke T cell products is a major barrier to the broader application of this approach. We are investigating a novel strategy to enable "off-the-shelf"' therapy with mismatched donor CAR19 T cells. Transcription activator-like effector nucleases (TALEN)s can be used to overcome HLA barriers by eliminating the risk of graft-versus-host disease (GvHD) through disruption of T cell receptor expression, and by simultaneously targeting CD52, cells can be rendered insensitive to the lymphodepleting agent Alemtuzumab. Administration of Alemtuzumab can then be exploited to prevent host-mediated rejection of HLA mismatched CAR19 T cells. We manufactured a bank of such cells from volunteer donor T cells under GMP conditions on behalf of Cellectis S.A for final stage validation studies using a third generation self inactivating lentiviral vector encoding a 4g7 CAR19 (CD19 scFv- 41BB- CD3ζ) linked to RQR8, an abbreviated sort/suicide gene encoding both CD34 and CD20 epitopes. Cells were then electroporated with two pairs of TALEN mRNA for multiplex targeting of both the T cell receptor alpha constant chain locus, and the CD52 gene locus. Following ex-vivo expansion, cells still expressing TCR were depleted using CliniMacs alpha/beta TCR depletion, yielding a T cell product with

Description: Chromosomal translocations involving the Mixed-Lineage-Leukemia (MLL) gene on chromosome 11q23 are frequent in infant acute leukemia and give rise to the formation of MLL-fusion genes. Several studies have addressed the importance of MLL-fusion activity for the initiation and maintenance of hematopoietic transformation. However, the dependence of established leukemias on MLL-fusion activity has not been previously addressed. We have developed a model for conditional expression of MLL-ENL in hematopoietic progenitor cells, in which expression of the fusion oncogene is turned off by doxycycline. In this study, immortalized myeloid cells conditionally or constitutively expressing the MLL-ENL fusion gene were used to induce acute myeloid leukemia (AML) in vivo. Primary recipients developed AML with a mean latency of 81.4 (±4.8) days. Secondary recipients developed AML with much shorter latencies than primary recipients regardless of whether the leukemic cells were freshly transplanted (26.8 (±6.8) days) or cultured in vitro for one month prior to transplantation (18 (±3.9) days). Genetic analysis revealed that some leukemic cells had acquired gross chromosomal abnormalities such as trisomy 6 or gains and losses of chromosome regions, which were not detected in the immortalised cells from which they were derived. Despite the acquisition of additional genetic abnormalities, the leukemic cells remained dependent upon MLL-ENL expression in vitro and in vivo. The leukemic cells terminally differentiated into neutrophils upon doxycycline treatment in vitro and established leukemias regressed following administration of doxycycline to recipient mice in their drinking water. Leukemic regression was accompanied by the complete loss of leukemic cells from the peripheral blood and differentiation of leukemic cells in the spleen. In 7 out of 34 doxycycline treated mice, remission was not sustained and the leukemias relapsed. However, most of these were shown to have acquired constitutive expression of MLL-ENL. This study demonstrates that leukemic cells are addicted to MLL-ENL expression and suggests that targeting the transcriptional/signalling networks established by MLL-fusion oncogenes in patients with 11q23 rearrangements would be a major therapeutic advance.

Description: Chromosomal translocations involving 11q23 are frequent in infant acute leukemia and give rise to the formation of MLL fusion genes. The mechanism of leukemic transformation by these fusions has been the subject of numerous investigations. However, the dependence of acute leukemia on MLL fusion activity in vivo and the efficacy of targeting this activity to eliminate disease have not been established. We have developed a model for conditional expression of MLL-ENL in hematopoietic progenitor cells, in which expression of the fusion oncogene is turned off by doxycycline. Conditionally immortalized myeloblast cells derived from these progenitors were found to induce leukemia in vivo. Leukemic cells isolated from primary recipient mice were shown to have acquired additional genetic abnormalities and, when transplanted into secondary recipients, induced leukemia with shortened latencies. However, the leukemic cells remained dependent on MLL-ENL expression in vitro and in vivo, and its ablation resulted in regression of established leukemias. This study demonstrates that even genetically complex leukemias can be reversed on inactivation of the initiating MLL fusion and has important implications for the design of novel leukemia therapies.