Clonal Evolution in a Murine CALM-AF10 Leukemia: Evidence of Functional Heterogeneity of Leukemia Stem Cells

Myeloid leukemia is caused by acquired genetic changes in haematopoietic stem cells. The combination of stepwise acquisition of genetic changes together with selection of the fittest clones results in great genetic and clonal heterogeneity. We used a CALM-AF10-driven retroviral transduction murine bone marrow transplantation leukemia model (MBMTLM) to study clonal hierarchy and clonal evolution starting with a primary leukaemia (Fig 1: Leu7) which developed after 131 days and had B220 marker expression on 4% of its cells. Limiting dilution assays (LDAs) showed that the leukemia stem cell (LSC) frequency of Leu7 was 1:2339 (95% confidence interval: 1:794-1:6885). Whole exome sequencing (WES) and analysis of the variant allele fraction of somatic mutations revealed that Leu7 was composed of a main clone (Fig 1: grey) with two subclones (blue and red). Half a million leukemic cells from Leu7 were transplanted into 4 sublethally irradiated recipients, which all developed secondary leukemias after a latency of 19 days (Leu7Sec1 to 4). All secondary leukemias showed similar B220 expression levels to Leu7, and all showed an expansion of the blue subclone. When again half a million cells each of one of the secondary leukemias (Leu7Sec2) were transplanted into 4 recipients, the expansion of the blue subclone continued, the red subclone vanished and, surprisingly, the proportion of B220 expressing cells increased to between 16 to 26%. LDAs showed that the LSC frequency of Leu7Sec2 had not changed. However, several of the leukemias from the LDAs had greatly varying latencies (27 to 193 days) and B220 marker expression (2 to 85%). Four of these tertiary LDA leukemias (Leu7Sec2Ter5 to 8), which each arose from a single LSC, were analysed more closely using WES. Leu7Sec2Ter5 showed a similar latency (27 days) and B220 expression levels like Leu7SecTer1 to 4 and also had the expansion of the blue subclone. Leu7Sec2Ter6 had a long latency of 69 days and a very low B220 expression. Leu7Sec2Ter6 was driven by a new, third subclone (pink), and both the blue and the red subclone disappeared. Very interestingly, Leu7Sec2Ter7 and Leu7Sec2Ter8 had a very long latency of 193 days, and showed an expansion of a subclone (green) of the red subclone. The B220 expression was high (37%) to very high (85%) in these two leukemias. Taken together, these observations paint an interesting picture with the blue subclone outcompeting the red subclone, as leukemias arising from the red subclone only appear after a long latency and in leukemias initiated by a single LSC, when there is no blue subclone LSC present. As the four leukemias (Leu7Sec2Ter5 to 8), which each were derived from a single LSC, showed striking differences in latency and surface marker expression, it can be concluded that this variation in phenotype is an intrinsic property of an individual LSCs most likely a consequence of the distinct combination of somatic mutations present in the individual LSCs. These observations also suggest that distinct LSCs with different properties might be present in a single human leukemia. Figure 1 Figure 1. Disclosures Browett: Janssen: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria.

Characterization of an Acute Myeloid Leukemia Murine Model Driven By MLL/AF9: Effect of Retroviral Insertion Sites and Somatic Mutations on Gene Expression

The MLL/AF9 fusion is found in approximately 30% of MLL-rearranged leukemias and has an intermediate prognosis. Genomically well-characterized murine leukemia models enable us to understand leukemogenesis. We generated a retroviral transduction murine bone marrow transplantation leukemia model (MBMTLM) using the MLL/AF9 fusion gene. Fifteen of 20 mice transplanted with syngeneic bone marrow transduced with a MLL/AF9 carrying retrovirus developed leukemia after a median latency of 149 days. Half a million leukemic bone marrow (LBM) cells from two of these primary leukemias, MA03-P and MA86-P, were transplanted into irradiated recipient mice to establish secondary leukemias, MA03-S (n=3) and MA86-S (n=4). Half a million LBM cells from these secondary leukemias were further transplanted into irradiated recipient mice to generate tertiary leukemias, MA03-T (n=3) and MA86-T (n=4). The latency of the leukemias shortened from 141 days in MA03-P to 18 and 22 days in MA03-S and MA03-T, respectively. Similarly, MA86-P had a latency of 98 days, and the latency was reduced to about 28 days in MA986-S and MA986-T. We used retroviral insertion sites (RISs) to track leukemia clones during serial transplantation. We identified 5 RISs in MA03-P. One RIS, RIS#1-03 at chromosome 7:4602500-4609499 accounted for 52.5% of the total RIS-related reads in MA03-P, while the other four RISs were each represented by fewer than 5% of the reads. Only RIS#1-03 was detected in all of the MA03 secondary and tertiary leukemias , indicating that the cells with RIS#1-03 were the dominant clone in MA03 leukemias. Two RISs were detected in MA86-P: RIS#1-86 at chromosome 19:41338500-41341999 and RIS#2-86 at chromosome 10:127106000-127109499 at 46.7% and 2.5%, respectively . RIS#1-986 was contained in the dominant clone as only this RIS was subsequently detected in the secondary and tertiary MA86 leukemias. The relatively long latency to leukemia development in our MLL/AF9 model was most likely due to the requirement of cooperating somatic mutations. We performed whole exome sequencing on DNA from LBM (n=15) and DNA from their corresponding germline (n=2). An average of 4.5 of single nucleotide variants (SNVs) and 11.4 indels affecting protein coding sequences were found in the MA03 family of leukemias (n=7) which, among others, mutated genes involved in tyrosine kinase pathways such as Epha5 and Pik3r1. We identified an average of 14.8 (SNVs) and 0.5 indels per exome in the MA86 leukemias (n=8). Transcription regulator (Brd1) and tumor suppressor genes (Stk11 and Trp53) were affected by somatic changes in the MA86 family. RNA sequencing was performed on LBM (n=15) and healthy bone marrow (HBM) (n=8). Principal component analysis (PCA) on the expression profiles showed that LBM samples clustered together. Differential gene expression analysis identified genes such as Six1, Eya1 and Bcor which had been reported in previous studies to be essential for leukemogenesis in MLL/AF9 murine model. We also observed downregulation of genes such as Gata2, Btg1, Ifitm1, which had been implicated in other types of leukemias. We next investigated the effect of the RISs and somatic mutations on gene expression. RIS#1-903 was in intron 1 of Ppp6r1. A reduction in fragments per kilobase of transcript per Million mapped reads (FPKM) of Ppp6r1 was observed in MA03 family leukemias compared to leukemias of the MA86 family which did not have RIS#1-03 and showed no difference to HBM samples (MA03: 87.71±1.5; MA86: 132.1±5.1; HBM: 77.56±1.7, p< 0.001). We then determined the expression of Tm9sf3 as it is located 600bp away from RIS#1-986. The FPKM of Tm9sf3 was significantly higher in LBM (both of MA903 and MA986 leukemias) than in HBM (LBM: 146.0±12.7; HBM: 64.66±2.8, p<0.001). In MA86 leukemias which all have RIS#1-86, the FPKM of Tm9sf was two fold higher than in MA03 leukemias without RIS#1-86 (MA86: 189.3±4.4; MA03:97.59±1.7, p < 0.001). In contrast, none of the somatic mutations had a significant effect on the expression of any of the mutated genes. In conclusion, we have established a MBMTLM driven by the MLL/AF9 fusion gene. This well-characterized model provides insights to further understand leukemia development and drug testing. Moreover, we demonstrated that RISs can have an impact on gene expression. Future work on whether Ppp6r1 and Tm9sf3 identified by our RIS analysis are drivers in MLL/AF9 leukemias is warranted. Disclosures Browett: MSD: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria.

Stem cell mutations can be detected in myeloma patients years before onset of secondary leukemias

Key Points Leukemia-associated mutations can be detected many years before the onset of secondary leukemias in myeloma patients. Stem and progenitor cells can act as reservoirs of mutations before the onset of secondary MDS and AML after treatment of myeloma.

Risk Of Acute and Chronic Leukemias Following Radiation Treatment For Locoregional Prostate Cancer In The United States Over 35-Years

Background Prostate cancer is the most common cancer diagnosis in men, and one of the leading indications for radiation therapy. The risk of resultant secondary leukemias has not been consistently established. We investigated the risk of all leukemias in a population-based cohort of patients (pts) with locoregional prostate cancer definitively treated with radiotherapy. Methods We queried the Surveillance, Epidemiology, and End Results (SEER) 17 registries to identify a cohort of men >20 years old (n = 183,268) with locoregional prostate adenocarcinoma newly diagnosed between January 1973 and December 2008. Pts who underwent initial treatment with radical prostatectomy (RP) were compared to pts receiving RP with external beam radiotherapy (RP w/EBRT) to investigate the risk of radiation-induced leukemias. These cohorts tend to be well matched regarding age, medical comorbidities and disease characteristics. All new leukemias occurring as a second primary cancer at least one year after the first diagnosis of prostate cancer were identified in SEER using the International Classification of Diseases for Oncology, Third Edition (ICD-O-3) morphology codes. Secondary leukemias included acute myeloid leukemia (AML); chronic myelogenous leukemia (CML); acute and chronic lymphocytic leukemia (ALL & CLL) and other categories as reported in SEER. Pts were observed from date of prostate cancer diagnosis until leukemia occurrence, death, or last date of follow-up. Univariate and multivariate analyses were performed using the Fine and Gray competing risk regression analysis with leukemia as a time-dependent endpoint and death from any cause or the diagnosis of any other second cancer as competing events. RP w/ EBRT group was compared with the RP cohort as the reference group, controlling for age. Hazard ratios (HR) with 95% confidence intervals (CIs) are reported. Results Median age was 67 years (yrs, range 22 – 105) at prostate cancer diagnosis: 67 yrs in RP and 68 yrs in RP w/ EBRT pts (p<0.0001); 158,913 (86.7%) were treated with RP and 24,355 (13.3%) with RP w/EBRT. Median follow-up was 7.6 yrs [(range, 1 – 35.5): 7.5 yrs in the RP group and 8.3 yrs in the RP w/ EBRT group, (p<0.0001)]. In total, 949 (0.5%) leukemia cases were identified: 336 (0.2%) acute leukemias [266 (0.2%) in the RP group and 70 (0.3%) in the RP w/ EBRT]; 538 (0.3%) chronic leukemias [462 (0.3%) in the RP group and 76 (0.3%) in the RP w/ EBRT] and 75 (0.04%) of unspecified histology. Histologic subtypes (per ICD-O-3 codes) were: AML (n=249), acute monocytic leukemia (n=18), ALL (n=24), other acute leukemias (n=45), other myeloid/monocytic/lymphocytic leukemias (n=48), aleukemic/subleukemic/NOS (n=27), CML (n=131) and CLL (n=407). Median age at acute leukemia diagnosis was 77 yrs [(range, 50 – 101): 78 yrs in the RP group and 76 yrs in RP w/EBRT pts, (p=0.0271)] and for chronic leukemias was 76 yrs [(range, 47 – 101): 76 yrs in the RP group and 77 yrs in the RP w/EBRT pts, (p=0.50)].The median time to develop acute leukemias was 6.0 yrs [(range, 1 – 28.2): 6.1 yrs in the RP group and 5.7 yrs in the RP w/EBRT pts, (p=0.20)] and chronic leukemias was 6.9 yrs [(range, 1 – 29.8): 6.7 yrs in the RP group and 8.6 yrs in the RP w/EBRT pts, (p=0.0020)]. The cumulative incidence rate (CIR) at 20 years for acute leukemias was 0.24% for the RP pts vs. 0.32% for the RP w/EBRT pts (p=0.0196). The CIR at 20 years for chronic leukemias was 0.47% for the RP pts vs. 0.36% for the RP w/EBRT pts (p=0.10). In univariate analyses, age >70 yrs (HR=1.40; CI, 1.13 – 1.74; p=0.0023), and those who received RP w/ EBRT (HR=1.49; CI, 1.14 – 1.94; p=0.0033) were significantly more likely to develop acute leukemias. In multivariate analysis, both advanced age (HR=1.40; CI, 1.13 – 1.74; p = 0.0023) and RP w/ EBRT (HR=1.49; CI, 1.14 – 1.94; p=0.0032), remained significantly associated with increased risk of acute leukemias. Radiation treatment was not significantly associated with the risk of developing chronic leukemias among pts treated with RP w/EBRT vs. RP [HR=0.91; CI, 0.72 – 1.16; p=0.45). Conclusions Among the best matched prostate cancer treatment cohorts, those who underwent EBRT following RP had a 49% increased risk of subsequent acute leukemias, although the absolute number of cases was low. Risk assessment in this cohort spans a time frame where radiation technologies have rapidly advanced and hence treatment period effects need to be considered in interpretation of results Disclosures: No relevant conflicts of interest to declare.

Outcome of Patients with Secondary Leukemias Managed with Different Treatment Strategies at a Single Center.

134 patients with a prior diagnosis of a malignant solid tumor presented between 1 Jan 95 and 31 Dec 04 to the leukemia service at PMH for management of a secondary hemopoietic malignancy or aplasia (AML n=99, MDS n=22, ALL n=11, AA n=2). The cohort included 65 males (median age 70 years, ranging from 18–94 years) and 69 females (median age 60 years, ranging from 25–87 years). The tumor types showed the expected gender based variation. Based on age, performance status and patient preference 81 individuals received remission induction therapy, while 53 patients were managed with supportive care. 49 of the patients undergoing induction therapy met the age criteria (70 years) for a blood and marrow transplant (BMT) and survived at least 70 days post initiation of induction therapy. 25 of the 49 patients underwent allogeneic BMT from related (n=20) or unrelated donors (n=5). The median survival of all 134 patients amounted to 314 days with an overall survival (OS) at 2 and 3 years of 25% and 20%. The respective values for patients undergoing induction therapy and supportive care were 413 days,36%,28% and 192 days, 9%,7% (p= 0.0002). Survival was strongly influenced by age (p= 0.0017), while gender (p=0.1436) and FAB subtype (p=0.219) did not contribute significantly to outcome. Subset analysis of the 25 BMT recipients showed a median survival of 922 days, with a 51% and 39.5 % OS at 2 and 3 years. The respective data for the remaining 24 not transplanted patients were not significantly different (p= 0.7508) with 522 days, 42% and 35%. Both subgroups however differed significantly in their causes of death favoring a lower relapse rate for transplant recipients and lower treatment related mortality (TRM) for non-transplanted patients (p=0.0012). In conclusion, this single center study confirms the relatively poor outcome for patients with secondary leukemias but is consistent with the view that patients able to undergo intensive therapy including transplantation may derive a significant survival benefit. The low relapse rate observed for BMT patients compared to patients not transplanted may translate into a survival benefit provided TRM can be reduced

Secondary Leukemias in Refractory Germ Cell Tumor Patients Undergoing Autologous Stem-Cell Transplantation Using High-Dose Etoposide

Purpose To quantify the risk of secondary leukemias in relapsed testicular cancer patients undergoing autologous stem-cell transplantation with high-dose etoposide. Patients and Methods Single institution, retrospective study of germ cell tumor patients who underwent autologous transplantation using high-dose etoposide from 1987 to 2001. Results One hundred thirteen patients received high-dose etoposide and carboplatin followed by autologous stem-cell transplantations for germ cell tumors. Follow-up ranged from 12 to 166 months (median, 51 months). Three patients (2.6%; 95% CI, 0.55% to 7.50%) subsequently developed leukemia at an average of 16 months post–autologous transplantation (range, 11 to 21 months). All three had received tandem transplantations and had been heavily pretreated, including at least one prior cycle of etoposide. Following autologous transplantation, all three patients exhibited refractory cytopenias before developing overt leukemia. All leukemias were of myeloid lineage. One patient developed an M2 with a t(8,21) chromosomal translocation; another, an M5 with a t(11,19); and one patient exhibited an unclassified leukemia with cytogenetic abnormalities resulting in monosomy for 7p and partial monosomy of 7q. Treatment of the leukemias involved allogeneic bone marrow transplantation. Conclusion High-dose chemotherapy using high-dose etoposide as therapy for relapsed germ cell tumors was associated with a 2.6% risk of developing a secondary myeloid leukemia. This figure was not significantly different from the expected rate of secondary leukemias when patients receive additional cycles of standard-dose etoposide as salvage chemotherapy for germ cell tumors. Other factors, including the use of platinum agents, may also have a role in leukemogenesis in this patient population.