ALBERT

Description: Abstract 2546 Introduction: We recently identified an alternative splice variant for IL23R (SV-IL23R), a type I cytokine receptor that regulates proliferation and differentiation via the JAK/STAT pathway. This SV-IL23R transcript contained a cryptic exon (CE) within intron 6 of the reference sequence and showed no evidence of expressing the proximal exons 1–6; rather, transcription of the SV-IL23R started with the CE, which introduced a novel start codon and maintained the normal reading frame through exons 7–11. As a result of these changes, majority of the critical extracellular binding structures of the receptor would be lost. Thorough review of databases identified previous reports of this transcript in melanoma and chronic myeloid leukemia, but studies have not systematically examined the expression of SV-IL23R in normal or malignant hematopoietic cells. Therefore, we performed a large retrospective study to examine the expression and clinical significance of SV-IL23R in a cohort of previously untreated pediatric AML patients. Methods: RNA from pretreatment bone marrow (BM) or peripheral blood (PB) was available in the Children's Oncology Group (COG) repository for 132 pediatric patients with AML (ages 0.1 – 21 yrs). Patients received double induction and intensification as per the single arm protocol COG-AAML03P1, which investigated the use gemtuzumab ozogamicin during induction and intensification. RNA was also available from 17 leukemic cell lines and the following subpopulations from healthy donors: mobilized PB CD34+ (N = 10), unsorted BM, (N = 10), unsorted PB (N =10), and T-cells (N = 10). SV-IL23R- specific primers were developed and optimized using standard RT-PCR conditions. The RT-PCR primers were developed to only amplify the SV-IL23R transcript. Results: We found no evidence of SV-IL23R expression in the 40 normal samples tested; however, SV-IL23R was expressed in 10 of the 17 leukemic cell lines. Of the 132 samples from pediatric patients, 57 (43%) expressed SV-IL23R. The presence of SV-IL23R expression was not significantly associated with age, gender, race, WBC count, blast percentage or FLT3-ITD status. However, the SV-IL23R expression was inversely correlated with low-risk t(8;21) or inv16 (Table 1). Moreover, patients with a CEPBA mutation were more likely to express SV-IL23R, while those with NPM1 or WT1 mutations were less likely to express SV-IL23R (Table 1). Interestingly, SV-IL23R expression was associated with an increased risk for resistant disease (29% vs. 14%, P = 0.039). Although there was a modest reduction in the estimated 5-year overall survival (OS, 62% vs. 72%, P = 0.19) and event free survival (EFS, 48% vs. 57%, P = 0.34), but these were not statistically significant (Table 1). Conclusions: This is the first study examining the prevalence and clinical significance of SV-IL23R in hematopoietic malignancies. We found that the blasts in 43% of evaluated pediatric AML patients expressed this transcript. Furthermore, SV-IL23R expression was significantly associated with resistant disease. Further studies examining samples from adult and additional pediatric AML patients are underway and will address the independent nature of this splice variant. If confirmed, SV-IL23R will become one of the most common biomarkers for AML and a potential target for future minimal residual disease assays. Moreover, its correlation with CR rates and potentially other clinical outcomes, if independent of other biomarkers, may enable us to add SV-IL23R to current portfolio of prognostic biomarkers, which may better enable us to more precisely risk-stratify future AML patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2520 INTRODUCTION. Age, cytogenetics, FLT3 and NPM1 mutations are the most significant prognostic factors (PFs) for adult AML treated with standard regimens, but the predictive significance of FLT3 and NPM1 with contemporary treatments is unknown. We examined the clinical significance of NPM1 and FLT3 mutations in adult de novo AML pts enrolled on SWOG study S0106. METHODS. S0106 was a randomized phase III clinical trial for pts of age 18–60 with de novo non-M3 AML, evaluating the effects of adding Gemtuzumab Ozogamicin (GO) to standard induction therapy (Cytosine Arabinoside and Daunomycin, AD), and of post-consolidation GO vs. no additional therapy (ASH, 2009, Abstract 790). Samples from 198 of the 600 eligible pts were evaluated. Analyses for nucleotide insertions in exon 12 of the NPM1 gene and internal tandem duplications (ITD) within exons 14–15 of FLT3 were performed using fragment analyses in diagnostic bone marrow (BM, N=190) and peripheral blood (PB, N=8) samples. Mutant/wild-type (WT) allelic ratios (AR) were computed for all mutations. Effects of mutations and other PFs on complete response (CR), resistant disease (RD), overall survival (OS) and relapse-free survival (RFS) were analyzed by logistic and Cox regression. P-values are 2-sided. RESULTS. Patient characteristics and outcomes are shown in Table 1. In univariate analyses, NPM1-Mut pts had significantly higher CR (81% vs. 58%, P=.0018) and lower RD (13% vs. 28%, P=.028) rates, better OS (64% vs. 47%, P=.045) and RFS (54% vs. 41%, P=.50). FLT3-ITD was not associated with CR or RD, but was associated with poorer OS (hazard ratio [HR] 2.28, P=.0011) and RFS (HR 2.74, P=.0009). FLT3-ITD length (range 18–366, median 46), FLT3 AR (range 0.18–8.2, median 0.98), and NPM1 AR (range 0.2–1.0, median 0.8) were not associated with CR, RD, or OS, but RFS tended to be lower with higher ITD length (P=.076). In multivariate analyses with other PFs, neither NPM1 nor FLT3 was associated with CR or RD rates, however the combined effects of FLT3 and NPM1 identified 3 mutation risk groups for OS (P=.0044, Fig 1A) and RFS (P=.0003, Fig 1B), since NPM1 did not significantly affect outcomes within the FLT3-ITD pts. These risk groups are FLT3-WT/NPM1-Mut (Good Risk: 3-yr OS 82%, RFS 69%), FLT3-WT/NPM1-WT (Intermediate Risk: OS 49%, RFS 43%), and FLT3-ITD (Poor Risk: OS 29%, RFS 14%). The impact of adding GO to induction therapy was examined within each risk group. In each risk group, CR rates were higher in the AD+GO arm, though not significantly so. Likewise, the RD rates were lower in the AD+GO arm, but this difference was significant only in the largest group: Intermediate Risk, FLT3-WT/NPM1-WT, 17% vs. 34% (P=.026). Treatment arm did not significantly affect OS and RFS in any mutation risk group. CONCLUSION. This study confirmed prognostic effects of FLT3 and NPM1 mutations in de novo AML pts treated with AD or AD+GO. Analyses of the joint impact of NPM1 and FLT3 mutations do not rule out the possibility that they act independently. With the small numbers of pts in the “good” and “poor” risk groups, there was no clear evidence that mutation status predicts clinical benefit from adding GO to therapy. We are evaluating additional samples and will update these results as data matures. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2550 Introduction: Aberrant expression of the wild-type transcript of interferon regulatory factor 8 (IRF8) in hematopoietic stem cells is associated with differentiation block, inhibition of apoptosis, and leukemogenesis. We recently identified 3 novel splice variants for IRF8 (IRF8-SVs). Our previous studies in adult AML patients found that IRF8-SVs were associated with a reduced complete remission (CR) rate and relapse free survival (RFS). Moreover, we showed the impact of RFS was independent of other known prognostic factors, such that all patients with ≥ 2 fold expression had relapsed or died within 1 year. To expand upon our previous studies, we examined the expression of IRF8-SVs in pediatric patients. Methods: RNA from pretreatment bone marrow (BM) or peripheral blood (PB) was available in the Children's Oncology Group (COG) repository for 206 pediatric patients with previously untreated AML (ages 0.1 – 21 yrs). Patients received double induction and intensification as per the single arm protocol COG-AAML03P1, which investigated the use gemtuzumab ozogamicin during induction and intensification. Quantitative RT-PCR (Q-RT-PCR) assay was developed to specifically quantify the collective expression of the three IRF8- SV transcripts. B-glucoronidase (GUSB), an endogenous control, was used to correct for RNA integrity. Fold change was computed relative to the pool of RNA from the peripheral blood of 10 healthy donors using the 2−ΔΔCtmethod. All Q-RT-PCR assays were performed in duplicate with appropriate negative and positive controls. Results: Increased IRF8-SVs expression (defined by 3 2-fold change) was present in 14% of the pediatric patients (29 of 206), which is similar to the 13% found in adult AML patients. Increased IRF8-SVs expression was not associated with gender, race, ethnicity, WBC count, blast percentage or mutations in FLT3, NPM1, WT1, or CEBPA. Patients harboring either t(8;21) or inv(16) were less likely to express increased levels of IRF8-SVs (P = 0.048 and P=0.016, respectively). There was no significant association between IRF8-SVs expression and other standard cytogenetic abnormalities. Importantly, pediatric AML patients with increased IRF8-SVs had a significantly worse 5-year event-free survival (EFS, 28% vs. 52%, P = 0.006) and overall survival (OS, 42% vs. 67%, P = 0.006, Figure 1). Conclusions: This is the first study examining the clinical significance of the novel IRF8 splice variants in pediatric patients with AML and follows upon our previous study examining IRF8-SVs in adult patients with AML. Overall, pediatric patients with AML displayed a similar prevalence for increased IRF8-SVs expression as their adult counterparts (14% vs. 13%, respectively). Likewise, the pediatric AML patients expressing increased IRF8-SV had significantly worse EFS. In addition, we also showed that increased expression of IRF8-SV was associated with a worse OS in the pediatric population, which was not appreciated in the adult study. Furthermore, we found a potential association between low-to-absent IRF8-SV expression and favorable cytogenetics [e.g., t(8;21), inv(16)]. This association with favorable cytogenetics may not have been appreciated in the previous adult study due to the small number of evaluated patients harboring favorable risk cytogenetic abnormalities. The present report confirms that the expression of IRF8-SVs is a novel AML prognostic biomarker, now demonstrating its clinical significance across patient populations of distinct age groups and those who received different treatment regimens. However, like many other biomarker studies (e.g., FLT3, NPM1, etc.), this study identified potential associations between IRF8-SVs and other established prognostic factors (e.g., CBF abnormalities). As such, additional studies examining larger numbers of adult and pediatric AML patients are underway. These ongoing studies will determine the optimal role of IRF8-SVs expression for risk-stratifying AML patients and how this novel prognostic biomarker can be incorporated into a more comprehensive risk-stratification scheme. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1679 Introduction: Aberrant expression of the wild-type transcript of interferon regulatory factor 8 (IRF8-WT) in hematopoietic stem cells is associated with differentiation block, inhibition of apoptosis, and leukemogenesis. We recently identified 3 novel transcript variants of IRF8 (IRF8-SVs). Each of the splice variants introduces a part of the terminal portion of intron 1 into the transcript, while splicing out exon 1 of the reference sequence (data submitted in a separate ASH abstract). Although these splice variants can be found at very low levels in hematopoietic cells from “healthy” adults, we have found that a subpopulation of AML patients expresses IRF8-SVs at much higher levels (〉2-fold) than hematopoietic cells from normal donors. Therefore, we performed a retrospective study to examine the prognostic significance of IRF8-SV transcripts in previously untreated adult AML patients. Methods: RNA from pretreatment bone marrow (BM) or peripheral blood (PB) was available in the Southwest Oncology Group (SWOG) repository for 194 patients with previously untreated AML. These patients received remission induction therapy with cytarabine and daunorubicin on SWOG trials S9031, S9126, S9333, or S9500. Compared to the other patients on these trials, the 194 patients had higher WBC counts (median 32,100/mcL vs. 7,300/mcL), BM and PB blast percentages (medians 70% vs. 60% and 44% vs. 20%, respectively) and were younger (median 61 vs. 66 years), more likely to have normal karyotype (44% vs. 34%), and less likely to have deletions of chromosomes 5q and/or 7q (11% vs. 25%). After adjusting for these factors, the 194 patients did not differ significantly in complete response (CR) or resistant disease (RD) rates, relapse-free survival (RFS), or overall survival (OS) from other trial participants. Quantitative RT/PCR (Q-RT/PCR) assays were developed to specifically quantify the collective expression of the three IRF8-SV transcripts. B-glucoronidase (GUSB) was used as endogenous control to correct for RNA integrity. Fold change was computed relative to the pool of RNA from the peripheral blood of 10 healthy donors using the 2-ΔΔCt method. All Q-RT/PCR assays were performed in duplicate with appropriate negative and positive controls. Geometric means of the duplicate fold-change values were used for all statistical analyses. Results: Increased IRF8-SVs expression (defined by 〉2-fold change) was present in 26 patients (13%) and was significantly associated with higher CD34 expression (P

Description: Introduction Younger acute myeloid leukemia (AML) patients with a NPM1 mutation but without FLT3-ITD (NPM1+/FLT3-ITD-) have favorable prognosis, but the prognostic significance of these mutations in patients older than 55 years is less clear. Therefore, we evaluated the prognostic impact of the NPM1+/FLT3-ITD- in older AML patients. Methods Samples were obtained from AML patients enrolled onto SWOG trials S9333, S9031, S9500 and S0106 who were ³55 years and received “7+3 like” induction regimens. Cytarabine/daunorubicin (S9031 and S9033) or HiDAC (9500 and S0106) were used for consolidation. Gemtuzumab ozogamicin, either during induction, consolidation, and/or post-consolidation, was administered to some patients enrolled onto S0106. Samples were examined for FLT3, NPM1, DNMT3A, IDH1/2 mutations using previously reported techniques. Results A total of 1,239 patients enrolled in these trials, and pre-treatment samples were available for 156 patients who were older than 55 years (median age of 60 years, range 55-83). NPM1 and FLT3-ITD mutations were present in 33% and 24% of the patients, respectively. The complete remission (CR) rate, 2-year overall survival (OS) and relapse-free survival (RFS) for the entire cohort were 62%, 31% and 32%, respectively. Increased age, unfavorable cytogenetics and FLT3-ITD were associated with a worse OS and RFS. NPM1 mutations were not significantly associated with OS or RFS. Patients were then divided into two age groups, 55-65 and 〉65 years, based on previous data that favorable prognostic factors such as good-risk cytogenetics have not been shown to be associated with favorable outcomes for AML patients age 〉65. Both age groups displayed similar patient characteristics (in regards to white blood cell count, bone marrow blast %, cytogenetics, secondary AML, sex and ECOG performance status), with the exception that 〉65 years group did not include any patients from S0106 or S9500 due to age restrictions for these trials. Patients 〉65 had a higher relapse rate (P =0.001) and significantly worse OS (P65 years had such a uniformly poor prognosis that this mutation lost much of its prognostic significance. NPM1 mutations were not significantly associated with either OS or RFS in either age group. We then examined the most favorable NPM1+/FLT3-ITD- genotype, which is currently being used to risk-stratify AML patients. NPM1+/FLT3-ITD- was associated with an improved OS in the 55-65 age group (P65 years with the NPM1+/FLT3-ITD- had a low CR rate and high 1-year relapse rate, which translated into a relatively poor 5-year OS (65 without this genotype (P=0.33). Since mutations in DNMT3A, IDH1/2 have been associated with adverse outcomes for patients with NPM1 mutations, samples with the NPM1+/FLT3-ITD- genotype were examined for these mutations. The frequencies for DNMT3A, IDH1/2 mutations were similar in both age groups, indicating that these mutations were not responsible for the age-dependent findings. Furthermore, multivariate models adjusting for known prognostic covariates showed that the NPM1+/FLT3-ITD- remained independently associated with improved OS for patients age 55-65 but not those 〉65. Conclusions This study represents one of the largest investigations on the prognostic significance of NPM1+/FLT3-ITD- in older AML patients. The NPM1+/FLT3-ITD- genotype remains a favorable-risk factor for AML patients age 55-65 years but may not be a favorable-risk factor for patients 〉65 years, at least not for those treated with standard induction followed by conventional consolidation. Disclosures: No relevant conflicts of interest to declare.

Description: INTRODUCTION. Most studies of AML biomarkers have examined cryopreserved mononuclear cells (MNCs) obtained from repositories. These MNCs typically include non-leukemic cells (e.g., lymphocytes) and heterogeneous populations of viable and nonviable AML blasts at various stages of differentiation. We hypothesize that these variables negatively impact the prognostic power of current biomarkers. In an attempt to improve the prognostic power of such biomarkers, we examined enriched populations of viable AML blasts from 190 randomly selected AML patients on SWOG trials. To our knowledge, this study represents the largest examination of the quality of cryopreserved AML blasts and the potential prognostic benefit of enriching for viable leukemic blasts. METHODS. Cryopreserved bone marrow (BM, N=124) and peripheral blood (PB, N=116) samples from 190 AML patients on SWOG trials (SWOG-9031, SWOG-9333, S0106, and S0112) were randomly selected. The samples were thawed and sorted for viable AML blasts using a combination of fluorochrome-conjugated antibodies and DAPI. DNA and RNA were extracted and evaluated from both the unsorted AML samples (A-MNCs) and viable AML blasts (A-Blasts). FLT3-ITD allelic ratio (AR) was quantified by fragment analyses. Expression of 13 potentially prognostic transcripts was quantified via quantitative RT/PCR: BAALC, CCNA1, CEBPA, ERG1, EVI1, FLT3, GATA2, IL3RA, JAG1, KIT, MN1, RUNX1, and WT1. Fold expression differences were computed by the comparative CT method. Associations between quantitative biomarkers (FLT3-ITD AR and transcript expression) and survival (overall, OS and relapse-free, RFS) were analyzed using Cox proportional hazards regression, with interaction terms for biomarker by cell population (A-MNCs vs. A-Blasts) or CD34 expression (separately for A-MNCs and A-Blasts). A predetermined FLT3-ITD AR cutoff of 0.5 was utilized based on results by Schneider et al. (Blood, 2012) and others. For paired samples, BM was used. RESULTS. Lymphocyte percentage varied (median 7.1%, range 0.4 - 70.1%) and was correlated with patients' pre-treatment blast percentages (rs = -0.29, P = 0.0002). Viability by DAPI varied widely (median 66.5%, range 5.2 - 95.6%), and AML blasts displayed inter- and intra-sample immunophenotypic heterogeneity, with the slight majority expressing CD34 (54%) using a predetermined immunofluorescence cut-off 〉 104. For OS, there was no significant difference (P=0.67) in the effect of FLT3-ITD AR between A-Blasts (HR=0.91, P=0.78) and A-MNCs (HR=1.05, P=0.88). However, for RFS, the effect of FLT3/ITD AR differed significantly (P=0.025) between A-Blasts (HR=1.93, P=0.14) and A-MNCs (HR=0.87, P=0.73). Kaplan-Meier curves of RFS by FLT3-ITD AR for A-MNCs (Figure 1A) and A-Blasts (Figure 1B) also suggest this trend. A similar, but nonsignificant, trend towards higher HRs in A-Blasts was displayed in analyses restricted to NPM1 mutated patients (Table 1). Similar analyses did not show such a striking interaction between cell population and transcript biomarkers, but we found that most transcripts displayed significant associations with immunophenotype (data not shown). Therefore, we examined if CD34 expression might impact the prognostic value of transcripts, separately by cell population. The expression of two genes (CCNA1 and GATA2) displayed significant interactions with CD34 expression in relation to clinical outcomes (Table 1). Moreover, the interaction between biomarker and CD34 expressions differed depending on which cell population was examined, such that the interaction was significant in the A-Blasts but not A-MNCs. CONCLUSION. Cryopreserved samples vary widely in percentages of non-leukemic, dying cells and differentiation stage of leukemic blasts. These factors impact the measurement of quantitative biomarkers and may also impact the significance and prognostic value of these biomarkers. Future studies must consider the effects that sample viability, composition, and differentiation stage may have on quantitative biomarkers. In addition, we are examining the potential impact that mutations in other genes (e.g., ASXL1, DNMT3A, RUNX1, etc.) may have on our results. ACKNOWLEDGEMENT. The authors wish to gratefully acknowledge the important contributions of the late Dr. Stephen H. Petersdorf to SWOG and to study S0106. SUPPORT. NIH/NCI grants CA160872,CA180819, CA180828, and CA180888. Disclosures Wood: Seattle Genetics: Honoraria, Other: Laboratory Services Agreement; Amgen: Honoraria, Other: Laboratory Services Agreement; Pfizer: Honoraria, Other: Laboratory Services Agreement; Juno: Other: Laboratory Services Agreement. Erba:Jannsen: Consultancy, Research Funding; Sunesis: Consultancy; Gylcomimetics: Other: DSMB; Agios: Research Funding; Celgene: Consultancy, Speakers Bureau; Seattle Genetics: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Incyte: Consultancy, DSMB, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy; Astellas: Research Funding; Millennium Pharmaceuticals, Inc.: Research Funding; Pfizer: Consultancy; Daiichi Sankyo: Consultancy; Celator: Research Funding; Juno: Research Funding. Othus:Celgene: Consultancy; Glycomimetics: Consultancy. Radich:Incyte: Consultancy; Novartis: Consultancy, Research Funding; Ariad: Consultancy; Gilliad: Consultancy.

Description: Abstract 1468 INTRODUCTION. Many molecular biomarkers have proven to be useful for guiding therapy for AML patients, yet remain inadequate for accurately predicting clinical outcomes. Possible explanations may be that certain biomarkers are more informative for restricted subpopulations of AML patients, or that the results of biomarkers must be interpreted in combination. Another explanation may be that there are methodological limitations in the assays. Most AML biomarkers are currently examined in MNCs, which include non-leukemic cells (lymphocytes, monocytes, and erythroblasts) and leukemic blasts. In addition, cells within the AML blast population display a wide spectrum of differentiation stages. To evaluate the potential impact of the non-leukemic cells in AML samples on the expression of prognostic transcriptional biomarkers, we examined enriched populations of cells from diagnostic AML samples. METHODS. Cryopreserved bone marrow (BM) and peripheral blood (PB) MNCs were obtained from 12 AML patients from the Leukemia Repository at the FHCRC. The cells were thawed and an aliquot was saved for future studies. Remaining cells were sorted for viable AML blasts using a combination of fluorochrome-conjugated antibodies (CD45, CD34, CD38, HLA-DR, and/or CD117) and DAPI. Additional sorts further differentiated the AML blasts into more or less mature (N=4 patients). RNA was extracted using Qiagen AllPrep RNA/DNA kit and reverse transcribed using AMV RT (Invitrogen). Expression of 22 potential prognostic biomarkers was evaluated using commercially available assays (Applied Biosystems) on ABI HT 7900 Fast Real-time PCR system. Fold differences in expression were computed by the comparative CT method, using GUSB to correct for RNA integrity and a pool of RNA from 10 PB from normal donors as a calibrator. RESULTS. Analyses showed marked differences between expression in the unsorted MNCs and AML blasts. Transcript expression was generally the same or more pronounced in AML blasts than in MNCs (e.g., BAALC, Figure 1A). In addition, any transcript differences identified in two populations generally diminished and were less dramatic in AML samples with high blast percentages. However, this was not the case for all samples and/or biomarkers. For example, CEBPA showed similar expression levels in MNCs and AML blasts. Attempts to adjust the signal in MNCs by blast percentages typically did not yield similar transcriptional levels for many samples. Similarly, the expression of examined prognostic biomarkers in subpopulations of AML blasts (mature vs. immature) showed a degree of heterogeneity among the patients. For some biomarkers, the immature population displayed a more pronounced fold difference in expression (e.g. BAALC, Figure1B), while for others, the expression was very similar in examined populations of most patients (e.g. CCNA1). CONCLUSION. As expected, the potential prognostic biomarkers demonstrated variable differences in expression in MNCs and enriched AML blasts, and these differences were even found to occur between the more and less differentiated AML blasts for the same patient. These differences may be of prognostic and predictive significance and may potentially impact the predictive ability of the biomarker. We are expanding these studies to include additional samples and determine if enriching for AML blasts, or even subpopulations within the blasts, may provide a more accurate assessment of prognosis. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3634 Introduction: Interferon regulatory factor 8 (IRF8) is a transcription factor that plays a critical role in normal hematopoiesis. IRF8−/− transgenic mice develop a myeloproliferative syndrome that transforms to acute myeloid leukemia (AML). IRF8 expression varies dramatically in the blasts of AML patients. A number of biological processes, including epigenetic changes, mutations, or alternative splicing, may contribute to the variability of IRF8 expression in AML patients. We investigated the potential causes of the aberrant IRF8 expression in AML blasts. Wild-type transcript (IRF8-WT): The entire coding sequence of IRF8 was amplified using HiFi Taq polymerase and sequenced from 7 leukemic cell lines and 12 AML samples. We did not find any mutations associated with aberrant expression of IRF8. Splice Variants (IRF8-SVs): The initial studies examining IRF8 coding region suggested transcript deviation in the 5′ region. The GeneRacer kit was used to sequence 5′-capped mRNA. We identified 3 previously-undescribed transcript variants. In all 3 sequences, exon 1 was spliced out and replaced by nucleotides from the terminal end of intron 1 (Figure 1A). Some of the splice variants contained potential in-frame start codons. Expression of 5′ Splice Variants in Leukemic Cell Lines and AML Samples: We examined the expression levels of transcript variants in 12 leukemic cell lines, 246 AML samples, and hematopoietic subpopulations of cells from “healthy” adults. IRF8-SVs were expressed at significantly higher levels in some AML blasts than normal hematopoietic cells. In fact, AML cell lines with the highest IRF8 levels primarily expressed the IRF8-SVs rather than the IRF8-WT. Promoter Methylation: Interferon response element (pIRE) within the IRF8 promoter, which controls the expression of the gene, was examined by PCR after bisulfite conversion. Cell lines with very low IRF8 expression were often methylated at the pIRE locus. In addition, some cell lines with marked over-expression of IFR8-SVs also displayed methylated pIRE, suggesting that promoter methylation may also be playing a role in controlling the expression of the splice variants. pIRE methylation was also examined in a more limited number of AML samples, finding that blasts with low expression of IRF8 were methylated at this locus (Figure 1B). Reversal of Splice Variant Expression Pattern: Our previous studies suggested that methylation may play a role in controlling IRF8-SVs expression. Therefore, we examined the effect of demethylating agents on IRF8-SVs expression in U937 cells – a leukemic cell line with very high levels of IRF8-SVs. IRF8 transcripts (WT and SVs) were examined before and after exposure to therapeutic levels of 5-azacytidine (5-Aza). These studies suggested that 5-Aza exposure and subsequent demethylation of the pIRE locus was associated with restoration of the IRF8-WT and decreased IRF8-SVs expression (Figure 1C). This later data provides additional evidence that pIRE hypermethylation may regulate the expression of the novel IRF8-SVs. Conclusions: We have identified novel IRF8 transcript variants that are over-expressed in AML blasts. These novel IRF8-SVs introduce additional nucleotides at the 5′ region of the gene and may result in a new start codon. AML blasts with high levels of IRF8 transcript often over-express IRF8-SVs. Although the functional significance of these variants is unknown, methylation may play a role in regulating their expression, such that demethylating agents decrease IRF8-SVs and promote the IRF8-WT expression. Additional studies are planned to investigate the biology and clinical significance of IRF8-SVs. Initial studies suggests that over-expression of IRF8-SVs is associated with an inferior clinical outcome for adult AML patients. Disclosures: No relevant conflicts of interest to declare.

Description: : INTRODUCTION: ELN-2017 guideline is the gold standard for risk stratifying AML patients. However, clinical prognostic factors such as age are not incorporated into the guideline. Therefore, we examined if novel prognostic models incorporating clinical factors and expression of select transcripts can improve the current guideline. The prognostic models were developed utilizing molecular data from unsorted mononuclear cells (MNCs) and viable leukemic blasts (VLBs). METHODS: Specimens were obtained from untreated AML patients (N=383) who received intensive chemotherapy on SWOG trials S9031, S9333, S0106 and S0112. Patients were randomly separated into discovery (N=190) and validation (N=193) cohorts. Participants provided informed consent in compliance with the Declaration of Helsinki, SWOG, NCI, and Fred Hutch regulations. VLBs were isolated using fluorescence-activated cell sorting (FACS, Pogosova et al., 2018, Biopreserv Biobank 16;42-52). RNA and DNA were extracted from MNCs and VLBs. FLT3-ITD, NPM1, CEBPA, ASXL1, RUNX1 and TP53 mutations were identified using fragment analyses and NGS. Transcript expression for BAALC, CEBPA, CCNA1, CD34, ERG1, EVI1, FLT3, GATA2, IL3RA, JAG1, KIT, MN1, RUNX1, and WT1 was quantified using Q-RT/PCR assays. Specimen attributes across paired samples were compared using McNemar's test and the Wilcoxon signed rank test. Associations with overall survival (OS), relapse-free survival (RFS), and complete remission (CR) were assessed using the Kaplan-Meier method, Cox proportional hazards regression, and logistic regression. Models were built in the discovery cohort using a pre-defined 3-step process of variable selection and 5-fold cross validation of this process; final model performance was evaluated in the validation cohort. RESULTS: Mutations, transcripts and clinical variables . Mutation frequencies, FLT3-ITD AR, and clinical outcomes were not significantly different between discovery and validation cohorts. The percentage of patients with high FLT3-ITD AR (per ELN-2017 definition) was increased in the VLBs compared to MNCs (77% vs 66%, respectively); FLT3-ITD AR was reclassified for 19 patients. Transcript expression was significantly increased in VLBs relative to MNCs for 8 transcripts (P

Description: Outcome of adult acute lymphoblastic leukemia (ALL) continues to be poor, and parameters to better discriminate patients with distinct prognosis are necessary. In studies comparing global gene expression differences between normal hematopoietic cells (whole bone marrow, peripheral blood, CD34+, and CD22+ sorted populations) and ALL cells using microarrays we found that the B-ALL cells showed evidence of increased expression of Connective Tissue Growth Factor (CTGF). The median log 2 transformed signal intensity of CTGF was 4.26 (range 3.95–4.76) in normal hematopoietic cells, and 6.59 (range: 3.85–10.62) in all leukemic samples; this difference in signal intensity is equivalent to a 5-fold increase in median expression of CTGF in leukemic cells. Therefore, we hypothesized that expression level of CTGF may have prognostic significance in adult ALL. Using real-time RT-PCR assays for CTGF we examined the expression of CTGF in 79 diagnostic ALL patients from SWOG protocol S9400 (28 bone marrow and 51 peripheral blood samples). Patients with L3 ALL were excluded from the study. The median age of patients was 35 (range 17–64), with the median WBC 23,400/ul (range 600–396,600), and peripheral blood blasts 56% (range: 0–98). Fifty patients had B-ALL (63%), 13 (16%) had T-ALL and lineage was unknown for 16 (20%). When treated as a continuous variable in a logistic regression model, the level of CTGF expression was significantly associated with inferior OS and DFS (p=0.007 and p=0.0012, respectively). When controlled for WBC and cell lineage, the association of CTGF with OS and DFS remained statistically significant. We then sub-grouped the ALL patients into three equal groups (tertiales) based on CTGF expression. This subgroup analysis found that the OS for patients in the highest tertile (highest CTGF expression) was approximately 11% (95% CI 0–24) at 5 years, as compared to 42% (95% CI 23–61) and 58% (95% CI 38–78) for patients with middle and low CTGF expression respectively (figure). In sub-analysis of patients with B-lineage ALL (n=50), the association of CTGF expression with OS and DFS was still statistically significant (p=0.009 and p=0.005) when treated as a continuous variable. This report is an example where a gene expression study detected a gene differentially expressed in leukemia, with clear clinical value. Moreover, this is the first report that correlates the level of expression of CTGF with outcome in ALL patients. We are actively pursuing the biological and clinical significance of CTGF in other ALL patients and model systems. Figure Figure