Presence Of Complex Karyotype Research Articles

Chemotherapy-Based Regimes Increase Karyotype Complexity of Del(11q) CLL Cells and Impact the Response to Subsequent Ibrutinib Treatment

The introduction of targeted agents in the treatment strategy of chronic lymphocytic leukemia (CLL) has changed the prognostic implications of some traditional high-risk markers such as del(11q). Recent data has suggested that this alteration might associate with superior progression-free survival (PFS) in patients (pts) treated with the BTK inhibitor ibrutinib (Ibr), especially in treatment-naïve (TN) cases (Kipps et al. 2019). This seems to be in marked contrast to data from chemotherapy (CHT) relapsed/refractory (CHT-R/R) pts treated with Ibr, where del(11q) cases showed inferior PFS than CLLs harboring other cytogenetic abnormalities (CAs) (excluding del(17p)) (Byrd et al. 2020). In this study we aimed to elucidate the mechanism underlying this differential response between TN and CHT-R/R del(11q) CLL pts treated with Ibr. We conducted a retrospective analysis of 211 CLL pts treated with Ibr-based regimes (n=88 TN; n=123 CHT-R/R). Median follow-up was 37 months (mo) [IQR 17-54] and CHT-R/R patients had a median of 3 therapies [1-7] (at least one line of CHT) prior to Ibr start. Thirty pts discontinued treatment due to adverse events. Restricting the analysis to the rest of the pts (n=181), the median PFS was significantly longer in del(11q) TN than del(11q) CHT-R/R cases (not reached (NR) vs 45 mo; P=0.001). TN del(11q) pts showed a 5-year PFS rate of 100% vs 75% of non-del(11q) TN pts (P=0.1). In contrast, the median PFS of del(11q) CHT-R/R pts was 45 vs 54 mo for non-del(11q) CHT-R/R cases (P=0.45). We next investigated whether differences at the genomic level would play a role in the diminished response of del(11q) CHT-R/R pts to Ibr. Targeted next-generation sequencing and miRNA profiling of del(11q) TN and CHT-R/R pts did not reveal substantial changes in the mutational and miRNA profile between both groups. In contrast, the presence of complex karyotype (≥3 CAs; CK) was able to stratify the outcome of del(11q) CHT-R/R pts (median 31 mo vs NR (CK vs non-CK); P=0.014). To validate these findings in an ex vivo setting, we co-cultured primary CLL cells from del(11q) TN and CHT-R/R pts (n=18) with HS5 stromal cells (+CpG and IL2) in the presence of Ibr. Consistent with the clinical findings, cells from del(11q) TN pts were more sensitive to Ibr than those cells from del(11q) CHT-R/R pts (P=0.012). Moreover, del(11q) CK CHT-R/R primary cells were more resistant to Ibr ex vivo than del(11q) non-CK CHT-R/R cells (n=8; P=0.05). To evaluate how CHT could impact the karyotype stability of del(11q) pts, we quantified the number of CAs in 263 CLL samples before (n=85) or after (n=178) treatment with CHT-based regimes and we found that the mean number of CAs was higher in del(11q) cases after treatment (mean 1.7 vs 3.7; P=0.01). Importantly, non-del(11q)/non-del(17p) pts did not show an increased number of CAs after CHT (mean 1.1 vs 1.0; P=0.36). In parallel, in a cohort of 314 CLLs treated with non-CHT agents (n=175 pre- and n=139 post-therapy), the mean number of CAs in del(11q) cases remained stable after treatment (mean 2.4 vs 1.9; P=0.1), suggesting that non-CHT regimes are less prone to induce karyotype instability in del(11q) CLL cells. We further analyzed longitudinal karyotype samples from del(11q) CLL pts before and after treatment with CHT (n=9) or non-CHT (n=19) agents. Notably, CHT induced karyotype changes in 6/9 del(11q) pts (P=0.03), whereas the vast majority (18/19) of del(11q) pts treated with non-CHT agents did not acquire additional CAs after treatment. Karyotype clonal evolution analyses revealed linear (3/6) and branched (3/6) patterns of evolution from the founder del(11q) clone in post-CHT samples, all resulting in del(11q) clones with a higher karyotype complexity, with a particular enrichment of chromosome 8 CAs. Conversely, karyotype clonal evolution was far less frequent (1/19) in del(11q) pts treated with non-CHT agents, even in the presence of multiple lines of therapy. Collectively, this work uncovers previously unknown differential Ibr sensitivity between del(11q) TN and del(11q) CHT-R/R CLLs and shows that such differential sensitivity could be mediated through CHT-induced karyotype complexity in the del(11q) clone of CHT-R/R del(11q) pts. Moreover, treatment with non-CHT agents maintains clonal stability of del(11q) cells, consistent with the prolonged PFS rates of del(11q) TN pts treated with Ibr. Thus, our data support the use of ibrutinib as a first-line therapy in del(11q) CLL.

AML-382 Cardiovascular Disease and Diabetes Mellitus Impact Outcomes of Patients With Acute Myeloid Leukemia

Incidence of both cardiovascular disease (CVD) and acute myeloid leukemia (AML) increases with age, and patients with AML more frequently have preexisting diabetes mellitus (DM), congestive heart failure, and myocardial infarction than non-cancer patients. We examined the influence of preexisting CVD and cardiovascular risk factors on the clinical outcomes of patients with AML. A total of 291 adult patients with AML with available smoking history treated at the University of Maryland Greenebaum Comprehensive Cancer Center 2014-2020 were reviewed retrospectively. Comorbidities identified through chart review were analyzed individually. Fisher's exact and t-tests were used to compare on-study characteristics and the outcomes of patients with and without CVD. Response rate (CR, CRi), overall survival (OS), and cumulative incidence of relapse were evaluated. OS was estimated by the Kaplan-Meier method. The multivariable logistic regression model was used to estimate the probability of achieving the best response. One-third of patients (34%) had preexisting CVD at AML diagnosis. Patients with CVD were more likely to have worse performance status and secondary AML (both P=0.03) and receive hypomethylating agent (HMA)-based induction therapy (72% vs. 38%, P<0.001). With a median follow-up of 40 months, CVD was associated with a lower probability of achieving CR or CRi (0.45 vs. 0.71, P<0.001). Having DM was independently associated with a worse response to treatment accounting for other on-study characteristics (OR=0.24, 95% CI: 0.08-0.80, P=0.01). Accounting for age, performance status, presence of complex karyotype, or secondary disease and treatment, CVD patients had worse OS than non-CVD patients (HR=1.5, 95% CI: 1.1-2.2, P=0.002), with 1- and 3-year OS of 44% versus 67% and 25% versus 40%, respectively (HR=1.9, 95% CI: 1.4-3.5, P<0.001), but no difference in cumulative incidence of relapse. Smoking did not impact OS (P=0.7). Sixty-two patients (21%) received allogeneic stem cell transplants, including 27% with preexisting CVD, and these patients had a worse OS (HR=2, 95% CI: 1-3.9). AML patients with CVD and those with DM have worse treatment outcomes and survival. Further clinical studies are required to understand the impact of CVD on the outcomes of AML patients.

Clinical Characteristics and Prognosis of Children with Myelodysplastic Syndrome

To analyze the clinical characteristics and prognosis of 40 children with myelodysplastic syndrome (MDS), and provide ideas for clinical diagnosis and treatment. The clinical characteristics, risk stratification, and different treatment regimens of 40 cases with MDS admitted in Department of Hematology of Children's Hospital of Soochow University from January 1, 2011 to December 31, 2017 was retrospectively analyzed. Kaplan-Meier survival curve were used to estimate 3-year overall survival (OS) rate and event-free survival (EFS) rate. In 40 cases, the ratio of male to female was 1.4∶1.0, male was more than female, and median age was 6.0 years old. Among them, refractory cytopenia (MDS-RCC) was the most common type, and 11 cases were chromosomal abnormalities, 21 cases genetic abnormalities. Fifteen cases accepted hematopoietic stem cell transplantation (HSCT) treatment, while 25 cases did not but drug therapy alone. The 3-year OS rate of the cases who accepted HSCT or not was (72.2±12.2)% and (35.3±10.2)% (P=0.039), 3-year EFS rate was (65.0±12.9)% and (19.2±8.4)% (P=0.012), respectively. Cox regression analysis showed that age < 7 years old (P=0.0333), initial diagnosed platelet < 50×109/L (P=0.007), presence of complex karyotypes and/or gene mutations (P=0.0002), and treatment without HSCT (P=0.016) were the high-risk factors of prognosis. All the children were classified according to IPSS, WPSS and IPSS-R, while analysis result showed that the above three risk assessment had limitations for risk assessment of MDS in children, they could not comprehensively assess the prognosis of children with MDS. MDS-RCC in children is more common. Cox multivariate analysis shows that age < 7 years old, initial diagnosed platelet < 50×109/L, presence of complex karyotypes and/or gene mutation, and treatment without HSCT are the high-risk factors of prognosis in children with MDS. HSCT is the most effective treatment to cure children with MDS at present. The current methods such as IPSS-R commonly used in assessment of prognosis in children with MDS show obvious limitation.

Post-relapse survival in Waldenstrom macroglobulinemia patients experiencing therapy failure following autologous transplantation.

Waldenström macroglobulinemia (WM) is a rare B-cell lymphoproliferative malignancy. Autologous hematopoietic cell transplantation (auto-HCT) is considered in a subset of WM patients with relapsed disease. While registry data has shown a benefit for auto-HCT in relapsed WM, there is a paucity of data on outcomes of patients relapsing after auto-HCT. Eligibility criteria included adult patients with relapsed/refractory WM who underwent auto-HCT between 2007 and 2017. The primary endpoint was post-relapse overall survival (PR-OS). Secondary endpoints were to identify factors prognostic of PR-OS. Of the 48 patients with WM who underwent auto-HCT, 22 (46%) experienced relapse following auto-HCT. Median PR-OS of relapsed WM patients after auto-HCT (n=22) was not reached (NR) (95% confidence interval [CI]: 17.5months-NR). Among patients who relapsed <1year versus ≥1year from auto-HCT, the median PR-OS was 18.4months (95%CI: 0.8-NR) months and NR (95%CI: 17.5-NR), respectively (p=0.06). Of note, disease status at the time of transplant, CR/VGPR versus partial remission did not appear to impact PR-OS. The median PR-OS was significantly longer in patients who received ibrutinib in the post-transplant setting compared to those who did not (NR vs. 18.4 months, 95%CI: 9.1-NR, p=0.02). On univariable analysis, the presence of complex karyotype (RR=4.87, 95% CI=1.22-19.53) and a higher number of prior lines of therapy (RR=1.81, 95% CI=1.23-2.67) were associated with a significantly higher risk of relapse. This is the only study to date that evaluated outcomes of WM patients who relapsed following auto-HCT and provides a benchmark for future trials evaluating survival following auto-HCT relapse.

Minority Health Disparities in Acute Myeloid Leukemia: A Metropolitan, Single-Center Retrospective Analysis

Background: Acute myeloid leukemia (AML) is an aggressive bone marrow cancer affecting 20,000 adults in the United States yearly. Five-year relative survival remains poor at 29.5%, though this has been steadily increasing. There are no known differences in diagnostic rates between racial and ethnic groups. Previous work has shown being African American is an independent predictor of poorer survival - particularly in impoverished areas and those with Medicaid. We aimed to identify potential racial disparities amongst our AML population - with special attention to insurance coverage, access to care, disease biology, regimen selection, toxicities, referral for allogeneic hematopoietic stem cell transplant (alloHSCT), and overall survival with non-Hispanic Whites as a control.Methods: This study is a retrospective analysis of patients diagnosed with AML and treated at Virginia Commonwealth University/Massey Cancer Center identified by our data analytics core from June 2018 to December 2020. Data were extracted and manually verified from the electronic medical record into an AML database instrument created in RedCap. Race was determined by self-report. Statistical analysis was performed using GraphPad Prism. Descriptive and inferential statistics were performed with comparisons between groups using an unpaired t-test with Welch's correction or with Fischer's exact test. Overall survival rates were evaluated using Kaplan-Meier analyses and compared using log-rank test. The event date was death and patients were otherwise censored at the date of last contact.Results: Our cohort consisted of 160 patients: 26.3% African Americans, 68.8% Whites, 1.9% Hispanic/Latinos, 1.9% other or declined to state, and 1.3% were unknown. To analyze the impact of minority populations, Hispanic/Latino and “other” categories were combined with African American into a “Non-White” cohort (N = 48) and compared to the “White” cohort (N = 110). There was no baseline difference in age (p= 0.212), Charleson Comorbidity Index (CCI) at presentation (p = 0.692), or ECOG status (p = 0.920) at presentation (Table 1). Assessment of disease biology, including European Leukemia Network risk stratification (p = 0.507), presence of complex karyotype (p = 0.366) and presence of TP53 mutations (p = 0.776) did not detect a statistically significant difference between the two groups (Table 1). Choice of intensive (vs non-intensive) induction based on physician's discretion was also similar (62.5% in non-White, 68.2% in Caucasians, p = 0.583). Toxicity analysis such as ICU during induction (p = 0.519) and death within 60 days of induction (p = 0.8) showed no difference between groups. In parameters assessing access to care, non-Whites were more likely than Whites to have either Medicaid or no insurance coverage, opposed to private insurance or Medicare (p = 0.0166). Despite this, there was no difference in overall survival assessed by log-rank test (p = 0.068) across all cytogenetic cohorts or with respect to the adverse risk cohort (p = 0.143), though the non-White cohort had a mOS of 286 days (9.4 months) compared to 764 days (25.1 months) in the White cohort. Rates of being lost to follow up were not different between the two groups (p = 0.34). However, rates of alloHSCT were approaching significance with p = 0.0528 favoring Caucasians.Discussion: Our data suggest similar disease biology at presentation amongst racial and ethnic groups, as well as similar comorbidities, performance status at diagnosis, and choice of induction regimen. As previous research has shown, our minority cohort was more likely to have no insurance or Medicaid than the Caucasian population. However, this did not lead to a statistically significant overall survival difference. Rates of alloHSCT were approaching statistical significance between the groups. This suggests that improving access to transplant might be one of the more effective tools for improving outcomes in this group. Additionally, demographics in the Richmond metro area demonstrate a population of 47% African American and 48% Whites, whereas our data showed 26% African Americans and 69% Whites, with no known strong racial predilection of AML based on SEER data (46% vs 54%, respectively). Thus, concern remains that there may be a significant number of patients with AML who either do not seek care, or present in a condition where treatment is no longer possible. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Prognostic Significance of Monosomy 7 with and without Complex Karyotypes in Acute Myeloid Leukemia (AML): A Single Institutional Analysis

Background: Over the past few years, the presence of monosomies in AML has emerged as an important prognostic factor. The incidence of monosomal karyotype (MK) increases with age, from 4% in patients ≤ 30 years to 20% in those > 60 years. Studies show that autosomal monosomies such as monosomy 7 confer very poor prognosis with overall survival in the range of 0-3%. About 30% of patients with AML have unfavorable karyotype with a complete response rate of 50% and a 5-year overall survival of 10-20% when treated with conventional chemotherapy. Of these unfavorable subgroup, about half will present with a complex karyotype (CK), which is associated with a poor outcome, and 40% of those with CK will have a MK which carries an even worse prognosis. In a single institutional retrospective study, we analyzed the prognostic significance of monosomy 7 with/without complex karyotype.Method: Cytogenetic and overall survival were analyzed in 137 patients with median age 65 (21-91). The analysis encompasses patients at Beaumont Hospital with confirmed AML treated between years 2010-2015 who had monosomy 7 with/without complex karyotype. Statistical analysis was performed by an independent investigator and overall Survival (OS) assessed by Kaplan-Meier method.Results: Out of 137 patients studied, we identified 99 patients with cytogenetic abnormalities. Among the latter subgroup, 16 (16.2%) patients had monosomy 7 with one patient <30 years old. While 3 (18.8%) patients with monosomy 7 also had core binding factor (CBF), 10 (62.5%) patients had monosomy 7 in the presence of CK and 5 (31.2%) patients had monosomy 7 without CK. Twenty-two (22.2%) patients had CK without monosomy 7. Statistical analysis using the SAS System for Windows version 9.3 showed that monosomy 7 in the absence of CK carries a worse prognosis than in the presence of CK (5-year OS, 0% and 6.3% respectively, p<0.002) (Table 1). The 5-year OS observed in patients with CK alone was 22.3% (Table 1). Thus, the prognostic impact of structural chromosomal abnormalities is intensified when they occur in association with a MK such as monosomy 7. Based on our analysis, MK is more predictive of poor outcome than CK and possibly other individual unfavorable karyotypes associated with very poor outcome.Conclusion: Occurrence of MK in the presence of CK is fairly common among patients diagnosed with AML and carries a worse prognosis. Although, our study represents a single institutional analysis, it concurs with recent reports on the prognostic significance of MK either alone or in combination with CK. Currently, the best chance at cure for patients with unfavorable karyotype who achieve compete response with induction therapy is allogeneic transplant. Such reports in our opinion should necessitate a more aggressive approach in terms of treatment options as well as early discussion with patients and families on the prognostic implications of these unfavorable karyotypes inorder to make more informed decisions. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Complex Karyotype Predicts the Prognostics Impact of WBC Count in Untreated Mantle Cell Lymphoma and Is Associated with Shortened Survival Independent of Specific Cytogenetic Abnormalities

Complex Karyotype Predicts the Prognostics Impact of WBC Count in Untreated Mantle Cell Lymphoma and Is Associated with Shortened Survival Independent of Specific Cytogenetic Abnormalities

Prediction of Treatment Efficacy in Relapsed Chronic Lymphocytic Leukemia

Background. The emergence of signaling pathway inhibitors (SPI) considerably improved the prognosis in relapsed chronic lymphocytic leukemia (R-CLL). Nevertheless, some patients cannot achieve optimal and sustained response. TP53 gene defects determine the refractoriness to immunochemotherapy (ICT) and lower rates of progression-free survival on SPI therapy. However, the prognostic value of complex karyotype (CK) in CLL has long been disputed. In recent years, greater attention has been placed on the prognostic impact of CK in the context of SPI therapy. Materials &amp; Methods. The study included 180 patients who received the drug treatment for R-CLL (113 of them with ICT, 67 of them with SPI). Their age at the onset of second-line therapy, the response to first-line therapy, early (&lt; 24 months) progression after first-line therapy, the number of therapy lines, and the presence of CK and TP53 gene defect were regarded as prognostic markers. Taking into account the clonal evolution in CLL, to assess the significance degree of the above predictors, Cox proportional hazards regression model with time-dependent variables was used. Results. The following independent factors proved to significantly reduce the risk of death: response achieved immediately after first-line therapy (hazard ratio [HR] 0.38; 95% confidence interval [95% CI] 0.20-0.72; p = 0.003) and the number of therapy lines (HR 0.56; 95% CI 0.37-0.86; p = 0.008). Treatment with only ICT in first and subsequent lines was associated with increasing risk of death (HR 2.25; 95% CI 1.09-4.63; p = 0.028). Genetic risks worsened the prognosis to a high degree of significance in the case of TP53 gene defect with excluded or unknown CK status (HR 10.54; 95% CI 4.25-26.17; p &lt; 0.001) as well as in the case of CK (HR 14.08; 95% CI 5.77-34.35; p &lt; 0.001). A significant predictor of poor outcome was reported to be the factor of unknown CK status without TP53 gene defect (HR 4.15; 95% CI 1.72-10.00; p = 0.002). Neither relapse time after first-line therapy nor the age &gt; 65 years showed independent prognostic value. Conclusion. Standard karyotyping of peripheral lymphocytes with specific stimulation establishes a clearer disease prognosis and suggests the optimal choice of R-CLL treatment strategy.

Characteristics and Outcomes of Therapy Related Myeloid Neoplasms in Patients with Multiple Myeloma Following Autologous Stem Cell Transplantation

Introduction: Patients who develop therapy-related myeloid neoplasms (t-MN) have dismal outcomes. Previous studies reported the incidence and risk factors associated with t-MN development. Lenalidomide, in the setting of oral, but not intravenous, melphalan is associated with a higher risk of t-MN (Palumbo et. al, Lancet Oncology, 2014). We carried out this study to evaluate the clinical and pathologic features of t-MN, therapies employed, and factors that predict long-term survival after diagnosis. Patients and methods: We identified patients who received the first ASCT 1998-2016 at our institution. t-MN was defined per the WHO 2016 classification. Median overall survival (OS) was calculated from the time of t-MN diagnosis to last follow-up or death. Statistical analyses were performed using SAS (JMP v14.1) or GraphPad Prism (v7). Results: Out of 2115 patients that underwent at least one ASCT, 53 (2.5%) developed t-MN. Thirty-five of 53 (66%) patients who developed t-MN had received lenalidomide. Among 2062 patients that did not develop t-MN, 916 (44.4%) patients received lenalidomide. Lenalidomide exposure was associated with development of t-MN (χ2 with Yate's correction 8.9, p=0.003). Ten patients were excluded from further analyses due to lack of follow up. Clinical characteristics are shown in Table 1a (N=43). Median age at t-MN diagnosis was 70 years (range 44-79). Median time from ASCT to t-MN was 5 years (range 1-15). After a median follow-up of 70 months (95% CI, 38-134), the median OS was 12 months (95% CI, 9-17, Figure). Primary causes of death were t-MN (71%), MM (12%), both (6%), and other including infection, GVHD, and unknown (12%). Seven (16%) had t-AML and 36(84%) had t-MDS. Three (42%) of 7 patients with t-AML had pure erythroid phenotype. At the time of last follow-up, 9 (21%) were alive. Seven (17%) underwent two ASCT, 16 (36%) received more than 2 years cumulative dose of lenalidomide. Median number of cycles of alkylator therapy including high-dose melphalan (HDM) used for ASCT was 2 (range 1-6). On univariate analysis, factors predicting OS from t-MN diagnosis were ≥ 2 alkylator vs. &lt; 2 cycles (11 vs. 27 months, p=0.02), ≥10% vs. &lt;10% blast at the time of t-MN diagnosis (5.5 vs. 17 months, p=0.01), and the presence of complex karyotype (CK) vs. not (11 vs. 17 months, p=0.03). There was no difference in survival among t-MDS patients who transformed to t-AML vs. those who did not, those who received 1 vs. 2 ASCT, and those with lenalidomide duration ≥ 2 vs. &lt;2 years. On multivariate analysis, the number of alkylator therapies and CK, but not % blasts predicted OS from t-MN diagnosis (Figure). Eight (19%) patients proceeded to receive an autologous (n=4) or allogeneic (n=4) transplant for t-MN (Table 1b). There was no survival advantage for patients who underwent SCT for t-MN, compared to those who did not (17 vs. 10 months, p=0.3). Similarly, there was no survival advantage for patients who received allo SCT compared to auto SCT (21 vs. 16 months, p=0.7). Survival for t-MDS and t-AML was also not different (15 vs. 6 months, p=0.2). There is only one long-term survivor in the transplant group (s/p allogeneic SCT). Four of seven (57%) patients died of progressive t-MN, 1 died of progressive MM, progressive MM and t-MN, and TRM each. Conclusions: Lenalidomide, in the setting of HDM, is associated with an increased risk of t-MN. Increased exposure to alkylators, a higher tumor burden, and the presence of CK predict poor survival in t-MN. As HDM/ASCT followed by lenalidomide maintenance remains the standard of care for eligible patients, minimizing exposure to other alkylator regimens may be prudent. While considered the standard, only a minority undergo transplant; and post-transplant outcomes remain poor. Studies to identify prognostic genetic and epigenetic risk factors are ongoing. Disclosures Al-Kali: Astex Pharmaceuticals, Inc.: Research Funding. Kumar:Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Research Funding. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Splenic Marginal Zone Lymphoma: Risk of Transformation, Clinico-Biological Features at Transformation and Prognosis

BACKGROUND: The clinical course of splenic marginal zone lymphoma (SMZL) is usually indolent, but development of transformation to aggressive lymphoma (SMZL-T) is seen in 10 to 15% of the cases. Risk factors to predict SMZL-T are poorly defined. AIM: The aims of our study were: 1.- to assess the risk of transformation in a large series of SMZL patients; 2.- to analyze the prognostic factors for this event and 3.- to analyze clinical and biological features of SMZL-T. PATIENT AND METHODS: We identified 84 SMZL diagnosed at the HCB between 1994 and 2017 and 15 of them (18%) had developed histological transformation (SMZL-T). In addition, we reviewed 21 SMZL with transformation referred to HCB from other centers. Median age at diagnosis of low-grade SMZL in the 84 patients was 63 years (range, 32-91), and male/female ratio was 34/50. Complex karyotype (CK) defined as ≥3 chromosomal aberrations by conventional cytogenetics was observed in 22% and del(7q) in 59% of cases. The risk distribution according to the Splenic Marginal Zone Lymphoma Study Group simplified score HPLL/ABC was: low: 34, intermediate: 51 and high: 15. After a median follow-up of 80 months (range, 1-265), 37 of 84 (44%) patients have died, with a median overall survival (OS) of 146 months. To assess the risk of transformation and to identify predictive factors for such event, we exclusively evaluated the cohort of 84 patients from HCB. RESULTS: Fifteen of the 84 patients in the HCB cohort (18%) developed histological transformation. The cumulative incidence of transformation at 60 months from diagnosis was 15% (95% CI: 7 - 23) (Figure). In univariate analysis, variables predicting transformation were anemia, lymphopenia, thrombocytopenia, hypoalbuminemia, CK and high-risk scores of Intergruppo Italiano Linfomi (IIL) and HPLL (p&lt;0.05 for all variables). In addition, lack of response to front-line treatment, relapse, and an early relapse at 12 and 24 months from treatment (p&lt;0.05 for all variables) predicted for a higher risk of transformation. In multivariate analysis, only CK maintained significance to predict transformation (HR 4.85 (CI 95%: 1.15-20.56) p=0.03). Main clinico-biological features of all 36 SMZL-T patients (including HCB patients and those referred from other centers) are described in the Table. Thirty-five patients transformed to diffuse large B-cell lymphoma (DLCBL) and 1 to Hodgkin lymphoma. Treatment at transformation is detailed in Table. Response was assessable in 34 cases: 21 (62%) achieved a complete response (CR), and 13 patients did not respond. Among responders, 11 continue in CR, 9 relapsed during the follow-up and 1 patient died from therapy related acute myeloid leukemia (AML) when still in CR. Twenty SMZL-T patients (56%) eventually died, 19 related to lymphoma and 1 from AML. The median survival of the 36 SMZL-T patients from the time of transformation (SFT) was 59 months; with 60-months SFT of 46% (95% CI: 28-64) (Figure). Variables at the time of transformation predicting survival were age &gt;60 years, thrombocytopenia and renal impairment. In addition, patients not achieving a CR, those not receiving an ASCT, and those who relapsed had a poorer SFT (p&lt;0.05 for all variables). In multivariate analysis the independent variables predicting a worse SFT were: age &gt; 60 years (HR 6.28 (95% CI: 1.55-25.39), p=0.01), thrombocytopenia (HR 32.77 (95% CI: 1.97-546.06), p=0.01), and response &lt; CR (HR 8.75 (95% CI: 2.76-27.72), p&lt;0.0001). Finally, SMZL-T was analyzed as a time-dependent variable for OS, showing that patients who eventually developed transformation had a significant higher risk of death (HR 3.894 (95% IC: 1.816-8.350), p&lt;0.001). CONCLUSION: The risk of SMZL-T in our series of HCB was 15% at 60 months. The only factor that significatively predicted risk of SMZL-T in a multivariant analysis was the presence of complex karyotype. SMZL-T patients had a significative shorter survival compared to not transformed patients. Disclosures Gine: Roche: Other: Travel expenses, Research Funding; Gilead: Other: Travel expenses, Research Funding; Janssen: Other: Travel expenses, Research Funding. Lopez-Guillermo:Celgene: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Janssen: Research Funding.

Complex karyotype in myelodysplastic syndromes: Diagnostic procedure and prognostic susceptibility.

Complex karyotype (CK) is a poor prognosis factor in hematological malignancies. Studies have shown that the presence of CK in myelodysplastic syndrome (MDS) can be associated with MDS progression to acute myeloid leukemia. The goal of this review was to examine the relationship between different types of CK with MDS, as well as its possible role in the deterioration and progression of MDS to leukemia. The content used in this paper has been obtained by a PubMed and Google Scholar search of English language papers (1975-2018) using the terms complex karyotype and myelodysplastic syndromes. A single independent abnormality can be associated with a good prognosis. However, the coexistence of a series of abnormalities can lead to CK, which is associated with the deterioration of MDS and its progression to leukemia. Therefore, CK may be referred to as a prognostic factor in MDS. The detection of independent cytogenetic disorders that altogether can result in CK could be used as a prognostic model for laboratory and clinical use.

TP53 Aberrations By FISH in CLL and Complex Karyotype at Transformation Predict for Worse Outcome in Diffuse Large B-Cell Lymphoma - Richter Transformation: A Single Institution Series of 75 DLBCL-RT Cases

Background:Richter transformation (RT), also known as Richter syndrome, is a rare complication of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) that has an aggressive clinical course and unfavorable prognosis. The majority of these transformations occur in the form of diffuse large B-cell lymphoma (DLBCL), and less frequently in the form of prolymphocytic leukemia (PLL), or classical Hodgkin lymphoma (cHL). We examined a series of RT cases with available CLL/SLL data diagnosed at our institution between 2000 and 2018 to identify clinical/biologic characteristics associated with adverse outcome.Design:After searching our pathology archives between the years 2000-2018, we identified a total of 83 RT cases: 75 DLBCL-RT (including 7 PLL-RT) and 8 cHL-RT. For the purposes of this study, we focused only on DLBCL-RT cases. All clinical, demographic, and pathologic data including cytogenetics (karyotype and FISH), immunohistochemistry (IHC), and flow cytometry were collected. Data points included age, gender, CLL/SLL histology (typical vs. atypical), CD5, CD38, ZAP-70 status, CLL karyotype, FISH [chromosomes 11q (ATM), trisomy 12, 13q, 14 (IGH), 17p(P53)]. In addition, time to transformation (months), site of transformation, transformation biopsy characteristics (germinal center vs. non-germinal center, CD5 status, along with MYC and BCL2 IHC status), and karyotype at transformation were collected. For the current analysis , karyotype and FISH data were dichotomized into (complex vs. non-complex) or (abnormality present vs. absent) respectively. Limited CLL treatment data was also collected but details on performance status, stage and IPI at transformation were not available.The clinical end point was overall survival (OS) determined as age at the time of transformation to death due to any cause, censoring patients without an event at last follow-up. Hazard ratios and 95% confidence intervals were derived from Cox proportional hazards regression models. Data analyses were performed using Stata®11.Results:Among DLBCL-RT patients, the median age at transformation was 66 years (range: 43-95). 87% of cases had antecedent CLL with a typical phenotype (CD5+, CD23+, FMC7-), 66% of CLL cases expressed CD38 (flow > 20% positivity) and 77% were positive for ZAP-70 (IHC). There was no correlation between CD38 expression and ZAP-70 (p=0.7). Fifty-three percent of the CLL cases harbored a complex karyotype. The most frequent abnormalities detected by FISH were TP53 aberrations (39%), followed by del13q (37%), Trisomy 12 (33%), IGH breaks (21%) and 11q/ATM deletion (17%).Transformation occurred at median of 59 months (0-352 range) after CLL diagnosis. DLBCL-RT was non-germinal center B-cell phenotype in 85% (n=27) of cases, with variable expression of CD5 (61%), p53 (50%), MYC (83%) and BCL2 (90%). EBER was infrequent at transformation (4%; n=23). Among DLBCL-RT, a total of 29 patients died at a median of 6 months (0-58 months) after RT diagnosis.In univariate analysis, age at transformation, type of CLL (typical vs. atypical), site (nodal vs. extranodal), CLL CD38 or ZAP-70 expression did not impact survival outcome. Only TP53 aberrations by FISH in antecendent CLL (p=0.02) (Figure 1) and the presence of complex karyotype at transformation were associated with adverse survival outcome (p=0.004) (Figure 2). MYC and/or BCL2 genetic alterations at transformation did not impact outcome. In a multivariable Cox model including both CLL TP53 aberrations and complex karyotype at transformation, only complex karyotype at transformation is weakly associated with outcome (p=0.09; hazard ratio 0.14 [0.015-1.36]; 95% CI) although this analysis is limited by the number of available patients with complete data (n=17).Conclusion:DLBCL-RT from an underlying CLL has a poor survival outcome and only TP53 alterations in the CLL and complex karyotype at transformation impacted survival. [Display omitted] DisclosuresStock:Jazz Pharmaceuticals: Consultancy. Riedell:Bayer: Consultancy, Speakers Bureau; Novartis: Consultancy; Kite Pharmaceuticals: Consultancy, Speakers Bureau. Smith:BMS: Consultancy; Portola: Honoraria.

Evolutionary Action Score of Missense TP53 Mutations Can Predict Outcome in Patients with Myelodysplastic Syndrome and Acute Myeloid Leukemia

Evolutionary Action Score of Missense TP53 Mutations Can Predict Outcome in Patients with Myelodysplastic Syndrome and Acute Myeloid Leukemia

Activation-induced deaminase and its splice variants associate with trisomy 12 in chronic lymphocytic leukemia

Activation-induced cytidine deaminase (AID) is a mutator enzyme essential for somatic hypermutation (SHM) and class switch recombination (CSR) during effective adaptive immune responses. Its aberrant expression and activity have been detected in lymphomas, leukemias, and solid tumors. In chronic lymphocytic leukemia (CLL) increased expression of alternatively spliced AID variants has been documented. We used real-time RT-PCR to quantify the expression of AID and its alternatively spliced transcripts (AIDΔE4a, AIDΔE4, AIDivs3, and AIDΔE3E4) in 149 CLL patients and correlated this expression to prognostic markers including recurrent chromosomal aberrations, the presence of complex karyotype, mutation status of the immunoglobulin heavy chain variable gene, and recurrent mutations. We report a previously unappreciated association between higher AID transcript levels and trisomy of chromosome 12. Functional analysis of AID splice variants revealed loss of their activity with respect to SHM, CSR, and induction of double-strand DNA breaks. In silico modeling provided insight into the molecular interactions and structural dynamics of wild-type AID and a shortened AID variant closely resembling AIDΔE4, confirming its loss-of-function phenotype.

Octamer-binding transcription factor 4 correlates with complex karyotype, FLT3-ITD mutation and poorer risk stratification, and predicts unfavourable prognosis in patients with acute myeloid leukaemia

ABSTRACTObjectiveTo investigate the correlation of octamer-binding transcription factor 4 (OCT4) expression with clinicopathological features and its predictive value for treatment response as well as survival profiles in de novo acute myeloid leukaemia (AML) patients.MethodOne hundred fifty-two de novo AML patients and 52 non-hematologic malignancy patients were recruited in this prospective cohort study. OCT4 expression was determined in bone marrow sample collected before treatment. Complete response (CR), event free survival (EFS) and overall survival (OS) were evaluated.ResultsCompared with the controls, OCT4 mRNA expression was higher in AML patients (P < .001), and higher OCT4 expression was correlated with presence of complex karyotype (CK) (P = .037), FLT3-ITD mutation (P = .012) and poorer risk stratification (P < .001) in AML patients. As to predictive value, OCT4 mRNA expression was decreased in patients achieved CR compared to non-CR patients (P = .022). Kaplan–Meier (K–M) curves showed that shorter EFS (9.0 (95% CI (7.7–10.3)) months vs. 25.0 (95% CI (17.5–32.5)) months, P < .001) and shorter OS (20.0 (95% CI (17.8–22.2) months vs. 33.0 months, P < .001) were observed in OCT4 mRNA high expression patients compared to OCT4 mRNA low expression patients. Multivariate Cox’s proportional hazards regression analyses revealed that OCT4 mRNA high expression was an independent predictive factor for shorter EFS and OS in AML patients.ConclusionOCT4 correlates with presence of CK, FLT3-ITD mutation and poorer risk stratification, and it could be served as a convincing biomarker for predicting unfavourable prognosis in AML patients.

The Impact of Clonal Dynamics on Prognosis and Outcome in Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a heterogeneous group of chronic myeloid neoplasms, in which disease progression is quite common, eventually terminating in secondary acute myeloid leukemia (sAML). To elucidate differential roles of mutations in MDS progression and sAML evolution, we investigated clonal dynamics of somatic mutations using targeted sequencing of 699 MDS patients, of which 122 were analyzed for longitudinally collected samples. Combining publicly available data, mutational data in a total of 2,250 MDS cases were assessed for their enrichment in specific disease subtypes. All samples were obtained after informed consent.Genotyping data from samples with low- (n=1,207) and high-risk (n=683) MDS as well as sAML (n=360) were available for most prevalently mutated 25 driver genes. In univariate comparison between low- and high-risk MDS, the majority of differentially mutated genes were enriched in high-risk MDS, except for SF3B1, which was more frequently mutated in low-risk MDS. Multivariate analysis was performed using a least absolute shrinkage and selection operator model. As a result, mutations in 7 genes (FLT3, PTPN11, WT1, IDH1, NPM1, IDH2,and NRAS) designated as 'Type-1' mutations, were significantly enriched in sAML compared to high-risk MDS. When comparison was made between high- and low-risk MDS, mutations in 10 genes, including GATA2, NRAS, KRAS, IDH2, TP53, RUNX1, STAG2, ASXL1, ZRSR2, and TET2, were enriched in high-risk MDS. The latter mutations are designated as 'Type-2' mutations, excluding NRAS and IDH2 mutations, which were already assigned to the Type-1 category.To characterize the chronological behavior of Type-1 and Type-2 mutations, we performed longitudinal analyses of 122 cases, of which 90 progressed to sAML. Overall, driver mutations tended to increase their clone sizes between two time points. In accordance with their significant enrichment in sAML, Type-1 mutations were more likely to be newly acquired at the second time points, compared to Type-2 and other mutations (P=0.0001). By contrast, in patients with high-risk MDS at the second time point, Type-2 mutations were more dominant than Type-1 mutations, and most of the Type-2 mutations (88%) increased their clone sizes at the second sampling. Similarly, Type-2 mutations found in high-risk MDS or sAML evolving from low-risk MDS increased their clone sizes more frequently (30 out of 38 mutations (79%)) than Type-2 mutations in stable low-risk MDS without disease progression over time (4 out of 11 (36%)) (P=0.02). These findings suggest that Type-1 and Type-2 mutations might be associated with progression from high-risk MDS to sAML and low- to high-risk MDS, respectively.To further clarify the effects of the different classes of mutations on progression to sAML, 429 patients with MDS were analyzed for progression free survival (or PFS). Patients with Type-1 mutations (Group-I) had a significantly shorter PFS, compared to those who had Type-2 mutations but lacked Type-1 mutations (Group-II) (HR=1.82, 95% CI:1.08−3.05; P=0.025). Nevertheless, PFS in Group-II cases was still significantly shorter than that in other cases (HR=2.46, 95% CI:1.43−4.23; P=0.001). Of note, some Group-II cases subsequently acquired Type-I mutations during progression to sAML. By contrast, SF3B1-mutated patients tended to show slower progression to sAML, unless they carried either of Type-1 or 2 mutations (Group-III). Finally, the effects of these mutations on overall survival (OS) were assessed in a larger cohort of patients with MDS (n=1,347). Group-I cases were shown to have a significantly shorter OS than Group-II cases (HR=1.50, 95% CI:1.20−1.86; P<0.001). Other independent prognostic factors included the International Prognostic Scoring System (IPSS) score and the mutational category (i.e., Group-I, -II, and -III) for PFS, while the presence of complex karyotypes, together with IPSS score, Group-I, -7/del(7q), age, and del(20q) were among the negative predictors of OS.In conclusion, our study has elucidated clonal dynamics associated with MDS progression and sAML evolution. Close monitoring of these sets of distinct mutations in the prospective fashion may help in the prediction of the clinical outcome in MDS. DisclosuresMakishima:The Yasuda Medical Foundation: Research Funding. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Ogawa:Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Activity of Hypomethylating Agents in the Treatment of Therapy-Related Myelodysplastic Syndrome

▪INTRODUCTION: Therapy-related myelodysplastic syndromes (t-MDS) are secondary malignancies that develop in patients who have received cytotoxic chemotherapy and/or radiation therapy for a primary cancer. t-MDS tend to have poor outcomes, with few available therapies and poor responses to conventional chemotherapies.PATIENTS AND METHODS: 263 patients diagnosed with t-MDS and treated with hypomethylating agents (HMA) between July 2001 and December 2015 at a single institution were retrospectively reviewed. Clinical and demographic data were obtained from clinical records. Response was assessed by modified 2006 International Working Group (IWG) criteria. Statistical analyses were performed with the IBM SPSS Statistics 23.0 software. Overall survival (OS) and leukemia free survival (LFS) were defined as the time between treatment onset and death or leukemia (or last contact), respectively. Data were summarized using median, range, and percentage. Censored endpoints were estimated by the nonparametric Kaplan-Meier method and compared using the log-rank test. All tests were 2-sided with significance set at p<0.05.RESULTS: Baseline characteristics are shown in Table 1. 56% of the patients were male. Median age at diagnosis was 66 years (range 13-87). According to the MDS International Prognostic Score System (IPSS), 17 patients (7%) belonged to the low risk group, 77 (29%) to the intermediate-1 risk group, 132 (50%) to the intermediate-2 risk group, and 34 (13%) to the high risk group. Following the Revised International Prognostic Score System (IPSS-R), 6 (2 %), 43 (16%), 20 (7%), 80 (30%), and 100 (38%) belonged to the very low, low, intermediate, high, and very high risk groups, respectively. Following the previously published scoring system for t-MDS, 57 (22%), 120 (46%), 63 (24%) belonged to the good, intermediate, and poor risk groups, respectively. The most common IPSS-R cytogenetic group was complex karyotype with more than 3 abnormalities, which was detected in 112 patients (43%), followed by the good risk group, which was present in 58 patients (22%). Next generation sequencing (NGS) analyzing a panel of 28 or 53 genes was available in 48 patients. The most frequent mutation observed was TP53 (present in 23 patients [48%]), followed by TET2 (present in 8 patients [7%]) and ASXL1 and DNMT3A (present in 3 patients each [6%]). Previous therapy received for the first neoplasm was chemotherapy alone in 123 patients (47%), radiotherapy alone in 27 (10%), or both in 111 patients (42%).A total of 73 (28%) patients received azacitidine in monotherapy (AZA) for the treatment of their t-MDS, 97 (37%) decitabine in monotherapy (DAC), and 92 (35%) were treated with combinations. Overall response rate (ORR) was 52% (137/263), including 33% (87/263) complete response (CR). No difference in terms of OS were observed between those patients treated with AZA vs DAC in monotherapy (p=0.75). The median duration of response was 6 months (range 1-69 months). Median OS was 13 months (Fig. 1A), and transformation to AML occurred in 126 patients (47.9%). There were statistically significant OS differences between responders and non-responders: 17 vs. 9 months, respectively (p<0.01) (Fig.1B).Univariate analysis revealed that concentration of hemoglobin (< 8 g/dl vs. ≥ 8 g/dl; p=0.003), number of platelets (<50x103/mm3 vs. ≥50x103/mm3; p< 0.001), presence of complex karyotype (p< 0.001), and presence of TP53 mutation (p=0.003) were all predictive of survival. After including all the significant factors in the univariate analysis in the multivariate model, only the presence of TP53 mutation was identified as independently associated with shorter survival in patients with t-MDS treated with HMA (HR[IC95%]: 3,44[1.53-7.34]) (Fig. 1C).CONCLUSION: Despite the poor prognosis of patients with t-MDS (median OS 12 months), HMA are clinically effective in the treatment of those patients, providing an ORR of 52% with a CR rate of 33%. Better OS were observed in responders when compared to non-responders, as well as in those patients without TP53 mutations. These results need to be confirmed in a larger numbers of patients. [Display omitted] [Display omitted] DisclosuresJabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Cortes:ARIAD: Consultancy, Research Funding; Bristol-Myers Squib: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding. DiNardo:Abbvie: Research Funding; Agios: Research Funding; Celgene: Research Funding; Daiichi Sankyo: Research Funding; Novartis: Research Funding. Konopleva:Calithera: Research Funding; Cellectis: Research Funding.

Abstract 2443: Ewing sarcoma progression associates with increasing chromosomal instability: A role for neuropeptide Y and its Y5 receptor

Abstract Ewing sarcoma (ES) is a tumor driven by EWS-ETS fusion proteins. Yet, the same fusions are present in localized and metastatic tumors that carry strikingly different prognoses. Despite low levels of genomic instability in primary ES tumors, the presence of complex karyotypes is one of a few adverse prognostic factors, implicating an acquired chromosomal instability (CIN) in ES progression. As transcriptional targets of EWS-ETS, neuropeptide Y (NPY) and its Y5 receptor (Y5R) are highly expressed in ES and further activated by hypoxia. We have found that overexpression of Y5R leads to defects in cytokinesis, followed by formation of polyploid cells, chromosome loss and CIN. Thus, the goal of our study was to determine whether CIN that is driven by hypoxia-induced activation of NPY/Y5R axis promotes ES metastases. ES cells were injected into gastrocnemius muscles of SCID/beige mice. Hypoxia in the resulting primary tumors was created by 72h ligation of the femoral artery. Then, the tumors were excised and mice were monitored for metastases. Tissues and cells derived from primary tumors and metastases were subjected to cytogenetic analyses. ES metastasis was associated with progressive genomic changes in tumor cells. Cells derived from primary tumors exhibited increases in nuclear sizes and ploidy, as compared to the original cells. Tumor hypoxia exacerbated this effect. This initial increase in ploidy was followed by a decrease in nuclear size, increase in mitotic errors and reduced chromosome numbers in cells from metastatic tissues, suggesting that ES progression associates with increased CIN and is triggered by cell polyploidization. This notion was confirmed by increased DNA copy number alterations in tissues from ES metastases observed in xenografts derived from 2 different cell lines and a clinical case of matched primary tumor and metastasis tissue (array-CGH). In SK-ES1 xenografts, these alterations involved gains in the locus of Y5R. Consequently, FISH identified an SK-ES1 clone with 3 copies of the Y5R gene. The percent of cells with Y5R gene amplification increased with the degree of SK-ES1 progression, with 16-24% cells in the original SK-ES1 cell line, 40-60% in primary tumors and 86-100% in metastases. This was associated with an increase in Y5R expression in metastatic tissues. Thus, the metastasis in SK-ES1 xenografts associated with a selection of the clone with amplified Y5R. SK-ES1 cells subjected to hypoxia in vitro presented with similar increases in nuclear sizes and enrichment in the clone with amplified Y5R (48%), as was observed in primary tumors. Y5R activation in normoxic SK-ES1 cells mimicked this effect. Our findings support the role for acquired CIN in ES progression and metastasis and implicate the hypoxia-induced activation of the NPY/Y5R axis as its potential trigger. Thus, Y5R antagonist may serve as an adjuvant treatment to prevent ES CIN and progression. Citation Format: Akanksha Mahajan, Sung-Hyeok Hong, Jason U. Tilan, Susana Galli, Congyi Lu, Jasmine Rodgers, Anju Duttargi, Rachel Acree, Luciane R. Cavalli, Joanna B. Kitlinska. Ewing sarcoma progression associates with increasing chromosomal instability: A role for neuropeptide Y and its Y5 receptor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2443.

Prognostic Impact of Molecular Mutations in Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS) on Allogeneic Hematopoietic Cell Transplant (HCT) Outcomes: Adverse Impact of TET2 Mutations

▪AML and MDS are heterogeneous myeloid neoplasms with variable biologic and clinical outcomes. Although allogeneic HCT is the only potentially curative therapy for high risk AML and MDS, survival after transplant remains poor, and identifying who benefits is challenging.We hypothesized that next-generation sequencing (NGS) mutational analyses can predict outcome in MDS and AML patients undergoing allogeneic HCT. We performed multi-amplicon targeted pre-HCT NGS using a somatic panel of the 60 most commonly mutated genes in myeloid neoplasias as previously determined by whole exome sequencing, on 123 patients with AML (N=64, 52%) and MDS (N=59, 48%) who subsequently underwent HCT. Median age at transplant was 53 years (range, 20-73). 21 (17%) patients had complex karyotype, 10 (8%) with monosomy 7, 48 (39%) normal, and 48 (39%) with other or unknown cytogenetic abnormalities. 45 (37%) patients were in a complete remission (CR) prior to transplant, while 78 (63%) were in less than a CR; with CR as defined by International Working Group criteria for MDS, or <5% blasts for AML. The majority of patients received myeloablative conditioning (N=83, 68%), and 40 (33%) received a reduced-intensity preparative regimen. Donor source was matched sibling (N=52, 42%), matched unrelated (N=56, 46%), cord-blood (N=12, 10%), and haplo-identical (N=3, 2%). Median follow up was 35 months (range 5-178). Mutations were analyzed individually and by molecular pathway. 88 (72%) patients had at least one mutation, most frequently in STAG2 (10.2%), TET2 (9.8%), ASXL1 (8.1%), and RUNX1 (8.1%). TP53 mutations were more common in MDS patients compared to AML (10% versus 1.6%, P=0.05). NRAS (P=0.019) and TP53 (P=0.022)mutations were more commonly associated with complex karyotype. Mutations in BCOR (P=0.048) and TP53 (P=0.047)were associated with less than CR, while TET2 (P=0.03)mutations were associated with CR prior to HCT. In univariable analyses, the presence of complex karyotype was associated with shorter overall (OS) and relapse-free survival (RFS) (hazard ratio [HR] 2.4; P=0.002 and HR 3.1; P<0.001). Mutations in TET2 (HR 2.1; P=0.042) and EZH2 (HR 2.3; P=0.048), or presence of any mutation in the histone modification pathway (ASXL1, EZH2, KDM6A, SUZ12); (HR 1.7; P=0.039) was associated with poor OS. The presence of any mutation in the DEAD box RNA-helicase family genes (DHX29, DDX54, DDX41) was associated with poor RFS (HR 3.1; P=0.009). Nothing except complex karyotype was specifically associated with higher relapse. Unlike in previous reports, TP53 mutations were not found to be significantly associated with poor OS or RFS, though these cases (N=7) were limited. In multivariable analyses, adjusting for clinical variables, complex karyotype remained significantly associated with poor OS (HR 2.7; P<0.001) and RFS (HR 3.9; P<0.001). TET2 also remained independently associated with poor OS (HR 2.4; P=0.022). Presence of any of the DNA methylation mutations (TET2, DNMT3A, IDH1, IDH2) was associated with poor RFS (HR 1.7; P=0.05). 3-year OS was 23% in patients with a complex karyotype versus 48% in patients without (P=0.002); and 14% in patients with a TET2 mutation and 46% without (P=0.042) (Figure 1).Molecular abnormalities are important variables in determining outcome after allogeneic HCT. We demonstrate that TET2 mutations in AML and MDS predict for poor survival after HCT. Ongoing serial mutational analyses in an extended cohort of patients will enhance our understanding of the role of NGS in informing care decisions for patients undergoing allogeneic HCT for AML and MDS. [Display omitted] DisclosuresMajhail:Gamida Cell Ltd.: Consultancy; Anthem Inc.: Consultancy. Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees.

Filanesib (ARRY-520) Demonstrates Potent and Rapid Activity in Preclinical Models of MM, Dependent on Bcl-2 Family Expression, and Synergistic with Dexamethasone and IMiDs

Abstract Introduction: Multiple myeloma (MM) is characterized by the presence of complex karyotypes and chromosome instability, suggesting that cell cycle checkpoints are defective. Filanesib (ARRY-520) is a highly selective, targeted inhibitor of kinesin spindle proteins (KSP), which are required to establish mitotic spindle bipolarity, driving centrosome separation. Filanesib monotherapy has demonstrated clinical activity in heavily pretreated MM patients (Lonial et al, ASH 2013). In this work we aimed to explore the preclinical activity of filanesib alone and in combination with IMiDs. Methods: In vitro activity of filanesib alone and in combination with IMiDs (thalidomide, lenalidomide and pomalidomide) was evaluated in MM cell lines by MTT assay and Annexin V, Propidium Iodide and DiOC6 analysis by flow cytometry, Western Blot and immunofluorescence. Synergy was quantified with combination indices (CI) by Calcusyn software. In vivo efficacy was assessed in a subcutaneous plasmacytoma model of MM1S in CB17-SCID mice. Results: Filanesib demonstrated significant activity in a broad panel of 11 MM cell lines, with 48-hour IC50 values ranging between 0.3 and 5 nM. Interestingly, the highest activity was observed in drug-resistant cell lines such as OPM-2 and RPMI-LR5. We next evaluated whether the cell death MoA was dependent on apoptosis or blockade of proliferation. Time response experiments performed in three different cell lines with different sensitivity to filanesib (OPM-2, MM1S and U266) showed accumulation of cells in G2/M, followed by loss of the mitochondrial membrane potential and activation of apoptosis. Accordingly, Western blot analysis demonstrated an activation of the mitotic checkpoint indicated by an increase in Cyclin B1, and activation of apoptosis with PARP, and caspase-3 and -7 cleavage. Furthermore, filanesib activity was very rapid as 15 minutes of exposure was sufficient to exert all of the apoptotic and cell cycle effects observed at 48 hours. Immunofluorescence microscopy using alpha-tubulin demonstrated that filanesib induces monopolar spindle formation. The sensitivity of MM to filanesib has been previously correlated with the cell-dependency of the anti-apoptotic protein Mcl-1. We, therefore, studied the basal levels of six Bcl-2 family members (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, Bad) in the 11 cell lines and observed a correlation between the basal levels of these proteins and drug sensitivity. In particular, we confirmed the relationship with the anti-apoptotic proteins Mcl-1 and Bcl-2 and demonstrated that conversely to what is observed with proteasome inhibitors, cells with high basal levels of pro-apoptotic Bax or Bak were more resistant to filanesib. Moreover, treatment with filanesib induced a clear decrease of Mcl-1 in the 3 cell lines analyzed (U266, MM1S and OPM-2) that coincided with a decrease of Bcl-2 in the most sensitive cell line, OPM-2. Finally, we evaluated the activity of filanesib in combination with IMiDs and dexamethasone. In vitro studies showed a synergistic effect of filanesib with dexamethasone (CI: 0.21), and with all IMiDs, being most pronounced with pomalidomide (CI: 0.09). Of note, this triple combination demonstrated the highest synergistic activity (CI: 0.06). These results were confirmed in vivo where the triple combination of filanesib, dexamethasone, and pomalidomide was also synergistic, with a significant reduction of tumor growth of up to 50 days, which correlated with a statistically significant survival improvement. Mechanistic studies on the combination are ongoing. Conclusions: Our results demonstrate the potent, rapid activity of filanesib which induces a cell cycle blockade through the inhibition of KSP, leading to apoptosis in MM. It was identified that this activity is dependent on the anti-apoptotic protein Mcl-1 and pro-apoptotic proteins Bax and Bak. Furthermore, in animal xenograft studies, filanesib exhibited robust synergism in combination with dexamethasone and any IMiD, pomalidomide being the most synergistic. These data are the rationale for the clinical trial Pomdefil which uses this combination in patients with relapsed refractory MM. The trial will begin soon as a collaboration of the Spanish Myeloma Group (GEM). This work was supported in part by Array BioPharma. Disclosures Humphries: Array Biopharma: Employment. Tunquist: Array Biopharma: Employment. Mateos: Array Biopharma: Honoraria. Ocio: Array Biopharma: Honoraria, Research Funding.