Neuberg, Donna

Abstract Background: Chronic lymphocytic leukemia (CLL) patients exhibit variable times from diagnosis to the need to initiate therapy. Previous studies have demonstrated that the rates of CLL cell proliferation (birth) and elimination (death plus recirculation to solid tissues) could be estimated using the incorporation of deuterium (2H), taken in the form of heavy water (2H2O), into the DNA of dividing cells. In a cross-sectional study of patients with early and late stage CLL, birth rates above 0.35% per day were associated with more aggressive disease. The NIH-sponsored CLL Research Consortium (CRC) conducted a prospective study on patients with early stage disease to assess the relationship between CLL-cell birth rate and the length of time to initiation of initial therapy. Methods: Eligible patients had untreated early stage (Rai 0, 1 or 2) disease, and were no more than 3 years from diagnosis. Additional clinical parameters included age at diagnosis, gender, mutational status of the immunoglobulin heavy chain variable region (IGHV), 70-kD zeta-associated protein (ZAP-70) (binary split at 20%) and CD38 (binary split at 30%) expression, and FISH cytogenetics by the Dohner hierarchy. Heavy water was administered daily for 6 weeks, and peripheral blood was obtained for kinetic measurements at the end of weeks 3, 6, 9, 12, and 16. CLL birth rates for blood samples were measured at the same facility (KineMed, Inc, Emeryville, CA) by quantifying deuterium incorporation into the deoxyribose moiety of DNA of tumor cells, using isotope ratio mass spectrometry (IRMS). Subjects were followed for time to administration of therapy; subjects still without treatment at time of analysis were censored, as were subjects who exited the study to receive treatment outside of the established criteria. To assess the impact of birth rate and other factors on time to first therapy, Kaplan-Meier curves were compared using the logrank test for univariable analyses; stepwise Cox proportional hazards regression models were used to build multivariable models. Results: Between July 2005 and February 2009, 119 subjects were enrolled through the CRC. 107 subjects were eligible. For 10 eligible patients, exit from the study prior to completion of protocol-mandated heavy water consumption (n=5) or technical issues (n=5) made it impossible to obtain estimates of birth rate. The 97 eligible subjects for whom birth rates were available form the basis of this report. 60% of subjects were male; median age 57 years (range 40-85). Median birth rate was 0.32%/day (range 0.07-1.31%/day). 43 subjects (44%) had birth rates higher than the previously reported threshold of 0.35%/day. With a median follow up of 4 years, 33 eligible subjects (34%) required treatment. IGHV status (31 subjects unmutated), elevated ZAP-70 (31 subjects), elevated CD38 (21 subjects), and hierarchical categorization of FISH anomalies (4 del(17p), 5 del(11q), 8 tri(12), 50 del(13q)) were examined for association with differential time to first therapy in univariable analyses. All were associated with earlier need for therapy (p <0.001) except for genomic aberrations defined by FISH (p = 0.96). Birth rate > 0.35%/day was also significantly associated with shorter time to first therapy (p < 0.001). In multivariable modeling, only birth rate and IGHV status met the criteria for inclusion (Wald p-value < 0.05). Hazard ratios were 3.51 (p = 0.002) for birth rate above 0.35%/day and 5.0 for unmutated IGHV status (p < 0.001). The association between elevated birth rate and shorter time to first treatment was observed within subjects with both unmutated and mutated IGHV (see Figure 1). Conclusion: An increased birth rate of CLL cells early in the disease course is a strong predictor of an earlier need for treatment in univariable analysis, as are unmutated IGHV, elevated ZAP-70 expression and elevated CD38 expression. However, only unmutated IGHV and an increased birth rate contribute significantly to the multivariable model. This adds a direct measure of B-cell kinetics to the array of biomarkers available to subjects with CLL. Figure 1 Time to treatment by birth rate and IGHV status Figure 1. Time to treatment by birth rate and IGHV status Disclosures Murphy: KineMed, Inc: Equity Ownership. Emson:KineMed, Inc.: Employment, Equity Ownership. Li:KineMed, Inc.: Employment, Equity Ownership. McConnel:KineMed, Inc.: Equity Ownership. Turner:KineMed, Inc.: Employment, Equity Ownership. Busch:KineMed, Inc.: Equity Ownership. Hellerstein:KineMed Inc: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Chiorazzi:KineMed, Inc.: Stock options Other.

Abstract Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease characterized by multiple low-frequency alterations including somatic mutations, copy number alterations (CNAs) and chromosomal rearrangements. We sought to identify previously unrecognized low-frequency genetic events, integrate recurrent alterations into comprehensive signatures and associate these signatures with clinical parameters. For these reasons, our multi-institutional international group assembled a cohort of 304 primary DLBCLs from newly diagnosed patients, 87% of whom were uniformly treated with state-of-the-art therapy (rituximab-containing CHOP regimen) and had long term followup. Tumors were subjected to whole exome sequencing with an extended bait set that included custom probes designed to capture recurrent chromosomal rearrangements. In this cohort, 47% of samples had available transcriptional profiling and assignment to associated disease subtypes. Analytical pipelines developed at the Broad Institute were used to detect mutations (MuTect), CNAs (Recapseq+Allelic Capseq) and chromosomal rearrangements (dRanger+Breakpointer) and assess clonality (Absolute). To analyze formalin-fixed paraffin-embedded tumors without paired normals we developed a method which utilized 8334 unrelated normal samples to stringently filter recurrent germline events and artifacts. Significant mutational drivers were identified using the MutSig2CV algorithm and recurrent CNAs were assessed with GISTIC2.0. In addition, we utilized a recently developed algorithm, CLUMPS2, to prioritize somatic mutations which cluster in 3-dimensional protein structure. With this approach, we identified > 90 recurrently mutated genes, 34 focal amplifications and 41 focal deletions, 20 arm-level events and > 200 chromosomal rearrangements in the DLBCL series. Of note, 33% of the mutational drivers were also perturbed by chromosomal rearrangements or CNAs, underscoring the importance of a comprehensive genetic analysis. In the large DLBCL series, we identified several previously unrecognized but potentially targetable alterations including mutations in NOTCH2 (8%) and TET2 (5%). The majority of identified chromosomal rearrangements involved translocations of potent regulatory regions to intact gene coding sequences. The most frequently rearrangements involved Ig regulatory elements which were translocated to BCL2, MYC, BCL6 and several additional genes with known roles in germinal center B-cell biology. After identifying recurrent somatic mutations, CNAs and chromosomal rearrangements, we performed hierarchical clustering and identified subsets of DLBCLs with comprehensive signatures comprised of specific alterations. A large subset of tumors shared recurrent alterations previously associated with follicular lymphoma including mutations of chromatin modifiers such as CREBBP, MLL2, and EZH2 in association with alterations of TNFRSF14 and GNA13 and translocations of BCL2. This cluster was enriched in GCB-type DLBCLs and contained a subset with select genetic alterations associated with an unfavorable outcome. An additional cohort of tumors was characterized by alterations perturbing B-cell differentiation including recurrent BCL6 translocations or alterations of PRDM1. A subset of these DLBCLs had alterations of NOTCH2 and additional pathway components or mutations of MYD88 in association with TNFAIP3, CD70 and EBF1, a master regulator of B-cell differentiation. An additional group of DLBCLs exhibited frequent MYD88 mutations in association with alterations of CD79B, PIM1, TBL1XR1 and ETV6 and BCL2 copy gain; these tumors were highly enriched for ABC-type DLBCLs. This coordinate signature and additional alterations of p53 pathway components were associated with outcome. We explored bases for the identified genetic alterations in DLBCL by performing an in silico mutational signature analysis. The most frequent mutational signatures were those of spontaneous deamination (aging) and AID with rare cases of microsatellite instability. We also assessed the clonality of identified genetic features to define cancer cell fraction and establish the timing of specific genetic events. The comprehensive genetic signatures of clinically annotated DLBCLs provide new insights regarding approaches to targeted therapy. Disclosures Link: Kite Pharma: Research Funding; Genentech: Consultancy, Research Funding. Rodig:Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding. Pfreundschuh:Boehringer Ingelheim, Celegene, Roche, Spectrum: Other: Advisory board; Roche: Honoraria; Amgen, Roche, Spectrum: Research Funding. Shipp:Gilead: Consultancy; Sanofi: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract Abstract 3865 ZAP-70 expression is a prognostic marker differentiating chronic lymphocytic leukemia (CLL) patients with indolent disease from those rapidly progressing after diagnosis and requiring treatment. We previously demonstrated in four independent clinical cohorts that DNA methylation at a single CpG dinucleotide in the ZAP-70 promoter region impacts on ZAP-70 transcriptional regulation and is prognostic in CLL (Claus et al. J Clin Oncol. 2012;30(20):2483–91). Here, we confirm and extend our previous findings by investigating the significance of ZAP-70 methylation relative to established prognostic biomarkers in CLL: IGHV mutation status or expression of ZAP-70 protein or CD38. DNA methylation analysis of the ZAP-70 core promoter region was conducted in mononuclear cells of 295 untreated CLL patients, previously reported by the CRC (Rassenti et al. Blood 2008;112:1923–30) using MALDI-TOF mass spectrometry (MassARRAY, Sequenom). The 295 patients had a median age of 55 years (range 26–82), 69% were male, and 46% were Rai stage 0, 47% stage I/II, and 7% stage III/IV at diagnosis. ZAP-70 protein and CD38 expression were measured by four-color flow cytometry and IGHV mutational status was assessed, as previously described. By hierarchical clustering, the previously identified single CpG dinucleotide (“CpG unit 1”, position +223 relative to the transcription start) separated patients with high methylation levels across the entire region from those with lower methylation levels at this specific site. With a median follow-up of 3.9 years in 50 patients who had not started treatment and 5.6 years among those still alive (n=196), patients with lower methylation levels at CpG unit 1 had shortened time to first treatment (TTT) and overall survival (OS) (both p<0.0001), confirming the prognostic relevance of this site. In our previous study, an optimal cut-off for CpG unit 1 methylation across four data sets was between 10% and 20%. In this cohort, the optimal cut-off across the two endpoints was between 15% and 25%. A natural gap in CpG unit 1 methylation levels occurred between 20% and 25%, adding potential biological significance, as lower values may represent patient cell samples in which both alleles are unmethylated. Applying a 20% cut-off, TTT and OS were significantly different for those with lower and higher methylation levels (both p<0.0001). The 5-year TTT estimates were 22% (95 % CI: 0.16–0.28) and 64% (95% CI: 0.52–0.74), respectively, for patients with low versus high methylation levels; 5-year OS estimates were 80% (95% CI: 0.73–0.85) and 90% (95% CI: 0.81–0.95). Low ZAP-70 methylation was significantly associated with CLL-cell expression of ZAP-70, CD38, or unmutated IGHV status (each p<0.0001). In a model of TTT, expression of ZAP-70 modified the strong effect of ZAP-70 methylation. In patients with CLL cells lacking ZAP-70 expression, high methylation was protective (hazard ratio [HR]=0.37, 95% CI: 0.23–0.58). In contrast, methylation had no significant effect in ZAP-70+ CLL (HR=1.47, 95% CI: 0.81–2.68); TTT was short regardless of methylation levels (Figure 1). IGHV mutation status and expression of CD38 did not provide significant additional prognostic information. The effect modification described for TTT was not observed for OS (p=0.57). Here, ZAP-70 methylation was the strongest risk factor (hazard ratio=0.32, 95% CI: 0.19–0.54; p<0.0001), and expression of ZAP-70, CD38 or unmutated IGHV did not significantly improve the ability to explain differences in OS. Hence, the prognostic value of ZAP-70 CpG unit 1 methylation was confirmed in a large patient cohort with extensive follow-up. We defined a clinically applicable threshold for ZAP-70 methylation assessment and determined its relative value to current prognostic markers in CLL. ZAP-70 methylation predicted longer TTT among patients with CLL cells lacking ZAP-70 expression, but not among those with ZAP-70+ CLL cells, although this could reflect sampling bias due to the small number of patients (n=16) in our cohort with both high methylation and expression of ZAP-70. Lastly, methylation was the strongest risk factor for OS, and neither IGHV mutation status nor expression of ZAP-70 or CD38 significantly improved the ability to predict prognosis. In conclusion, ZAP-70 DNA methylation represents a clinically valuable biomarker for predicting TTT and OS in previously untreated patients with CLL. Disclosures: Gribben: Gilead: Honoraria; Mundipharma: Honoraria; GSK: Honoraria; Pharmacyclics: Honoraria; Roche: Honoraria; Celgene: Honoraria. Kay:Celgene: Research Funding.

Key Points The expression level of ROR1 on CLL cells varies between patients. High-level CLL-cell expression of ROR1 associates with more aggressive disease.

SummaryThis study revisited the Dohner prognostic hierarchy in a cohort of 1585 well‐documented patients with chronic lymphocytic leukaemia. The duration of both time to first treatment (TTFT) and overall survival (OS) were significantly longer than observed previously, and this is at least partly due to improved therapeutic options. Deletion 13q remains the most favourable prognostic group with median TTFT and OS from fluorescence in situ hybridization (FISH) testing of 72 months and >12 years, respectively. Deletion 11q had the poorest median TTFT (22 months) and 17p deletion the poorest median OS (5 years). The percentages of abnormal nuclei were significantly associated with differential TTFT for the trisomy 12, 13q and 17p deletion cohorts but not for the 11q deletion cohort. From the date of the first FISH study, patients with >85% 13q deletion nuclei had a notably shorter TTFT (24 months). Patients with ≤20% 17p deletion nuclei had longer median TTFT and OS from the date of the first FISH study (44 months and 11 years), and were more likely to be IGHV mutated.

It is controversial whether acute myeloid leukemia (AML) patients with 20–29% bone marrow (BM) blasts, formerly referred to as refractory anemia with excess blasts in transformation (RAEBT), should be considered AML or myelodysplastic syndrome (MDS) for the purposes of treatment and prognostication. We retrospectively studied 571 de novo AML in patients aged >50 years, including 142 RAEBT and 429 with ≥30% blasts (AML30), as well as 151 patients with 10–19% BM blasts (RAEB2). RAEBT patients were older and had lower white blood count, but higher hemoglobin, platelet count, and karyotype risk scores compared to AML30, while these features were similar to RAEB2. FLT3 and NPM1 mutations and monocytic morphology occurred more commonly in AML30 than in RAEBT. RAEBT patients were treated less often with induction therapy than AML30, whereas allogeneic stem cell transplant frequency was similar. The median and 4‐year OS of RAEBT patients were longer than those of AML30 patients (20.5 vs 12.0 months and 28.6% vs 20.4%, respectively, P = 0.003); this difference in OS was manifested in patients in the intermediate UKMRC karyotype risk group, whereas OS of RAEBT patients and AML30 patients in the adverse karyotype risk group were not significantly different. Multivariable analysis showed that RAEBT (P < 0.0001), hemoglobin (P = 0.005), UKMRC karyotype risk group (P = 0.002), normal BM karyotype (P = 0.004), treatment with induction therapy (P < 0.0001), and stem cell transplant (P < 0.0001) were associated with longer OS. Our findings favor considering de novo RAEBT as a favorable prognostic subgroup of AML. Am. J. Hematol. 89:E193–E199, 2014. © 2014 Wiley Periodicals, Inc.

Abstract Genetic lesions such as BCR–ABL1, E2A–PBX1, and MLL rearrangements (MLLr) are associated with unfavorable outcomes in adult B-cell precursor acute lymphoblastic leukemia (B-ALL). Leukemia oncoproteins may directly or indirectly disrupt cytosine methylation patterning to mediate the malignant phenotype. We postulated that DNA methylation signatures in these aggressive B-ALLs would point toward disease mechanisms and useful biomarkers and therapeutic targets. We therefore conducted DNA methylation and gene expression profiling on a cohort of 215 adult patients with B-ALL enrolled in a single phase III clinical trial (ECOG E2993) and normal control B cells. In BCR–ABL1-positive B-ALLs, aberrant cytosine methylation patterning centered around a cytokine network defined by hypomethylation and overexpression of IL2RA(CD25). The E2993 trial clinical data showed that CD25 expression was strongly associated with a poor outcome in patients with ALL regardless of BCR–ABL1 status, suggesting CD25 as a novel prognostic biomarker for risk stratification in B-ALLs. In E2A–PBX1-positive B-ALLs, aberrant DNA methylation patterning was strongly associated with direct fusion protein binding as shown by the E2A–PBX1 chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq), suggesting that E2A–PBX1 fusion protein directly remodels the epigenome to impose an aggressive B-ALL phenotype. MLLr B-ALL featured prominent cytosine hypomethylation, which was linked with MLL fusion protein binding, H3K79 dimethylation, and transcriptional upregulation, affecting a set of known and newly identified MLL fusion direct targets with oncogenic activity such as FLT3 and BCL6. Notably, BCL6 blockade or loss of function suppressed proliferation and survival of MLLr leukemia cells, suggesting BCL6-targeted therapy as a new therapeutic strategy for MLLr B-ALLs. Significance: We conducted the first integrative epigenomic study in adult B-ALLs, as a correlative study to the ECOG E2993 phase III clinical trial. This study links for the first time the direct actions of oncogenic fusion proteins with disruption of epigenetic regulation mediated by cytosine methylation. We identify a novel clinically actionable biomarker in B-ALLs: IL2RA(CD25), which is linked with BCR–ABL1 and an inflammatory signaling network associated with chemotherapy resistance. We show that BCL6 is a novel MLL fusion protein target that is required to maintain the proliferation and survival of primary human adult MLLr cells and provide the basis for a clinical trial with BCL6 inhibitors for patients with MLLr. Cancer Discov; 2(11); 1004–23. ©2012 AACR. Read the Commentary on this article by Cimmino and Aifantis, p. 976. This article is highlighted in the In This Issue feature, p. 961

Abstract Chemotherapy-related myeloid neoplasia (t-MN) is a significant late toxicity concern after cancer therapy. In the randomized intergroup phase 3 E2997 trial, initial therapy of chronic lymphocytic leukemia with fludarabine plus cyclophosphamide (FC) compared with fludarabine alone yielded higher complete and overall response rates and longer progression-free, but not overall, survival. Here, we report t-MN incidence in 278 patients enrolled in E2997 with a median 6.4-year follow-up. Thirteen cases (4.7%) of t-MN occurred at a median of 5 years from initial therapy for chronic lymphocytic leukemia, 9 after FC and 4 after fludarabine alone. By cumulative incidence methodology, rates of t-MN at 7 years were 8.2% after FC and 4.6% after fludarabine alone (P = .09). Seven of the 9 cases of t-MN after FC occurred without additional therapy. Abnormalities involving chromosomes 5 or 7 were found in 10 cases, which suggests alkylator involvement. These data suggest that FC may induce more t-MN than fludarabine alone.

AbstractLeukemia-cell expression of ZAP-70, CD38, or unmutated immunoglobulin heavy chain variable region genes (U-IGHV) each is associated with aggressive disease in patients with chronic lymphocytic leukemia (CLL). To assess the relative strength of each marker, we defined thresholds for designating a case as positive for CD38 or ZAP-70 in a test cohort of 307 patients and used these data-defined criteria to stratify patients in an independent cohort of 705 patients. Multivariable analysis revealed that ZAP-70 was the strongest risk factor. Knowledge of the IGHV mutation status or CD38 did not improve our ability to predict the time to first treatment except for ZAP-70–negative cases, which could be segregated into 2 groups of intermediate-risk or low-risk disease based on whether they expressed unmutated or mutated IGHV. ZAP-70 maintained its high relative prognostic value for the subset of patients with early-stage, asymptomatic disease, including patients evaluated within 1 year of diagnosis. Although it is premature to recommend therapy based on these risk factors, patients with ZAP-70–positive CLL cells should be monitored closely for disease progression as they have a median time from diagnosis to requiring initial therapy by standard criteria of approximately 3 years.