Oakes, Christopher C.

Abstract High-count monoclonal B-cell lymphocytosis (HCMBL) is a precursor condition to chronic lymphocytic leukemia (CLL). We have shown that among individuals with HCMBL, the CLL-International Prognostic Index (CLL-IPI) is prognostic for time-to-first therapy (TTFT). Little is known about the prognostic impact of somatically mutated genes among individuals with HCMBL. We sequenced DNA from 371 individuals with HCMBL using a targeted sequencing panel of 59 recurrently mutated genes in CLL to identify high-impact mutations. We compared the sequencing results with that of our treatment-naïve CLL cohort (N = 855) and used Cox regression to estimate hazard ratios and 95% confidence intervals (CIs) for associations with TTFT. The frequencies of any mutated genes were lower in HCMBL (52%) than CLL (70%). At 10 years, 37% of individuals with HCMBL with any mutated gene had progressed requiring treatment compared with 10% among individuals with HCMBL with no mutations; this led to 5.4-fold shorter TTFT (95% CI, 2.6-11.0) among HCMBL with any mutated gene vs none, independent of CLL-IPI. When considering individuals with low risk of progression according to CLL-IPI, those with HCMBL with any mutations had 4.3-fold shorter TTFT (95% CI, 1.6-11.8) vs those with none. Finally, when considering both CLL-IPI and any mutated gene status, we observed individuals with HCMBL who were high risk for both prognostic factors had worse prognosis than patients with low-risk CLL (ie, 5-year progression rate of 32% vs 21%, respectively). Among HCMBL, the frequency of somatically mutated genes at diagnosis is lower than that of CLL. Accounting for both the number of mutated genes and CLL-IPI can identify individuals with HCMBL with more aggressive clinical course.

Abstract Monoclonal B-cell lymphocytosis (MBL) progresses to chronic lymphocytic leukemia (CLL) requiring therapy at 1% to 5% per year. Improved prediction of progression would greatly benefit individuals with MBL. Patients with CLL separate into 3 distinct epigenetic subtypes (epitypes) with high prognostic significance, and recently the intermediate epitype has been shown to be enriched for high-risk immunoglobulin lambda variable (IGLV) 3-21 rearrangements, impacting outcomes for these patients. Here, we employed this combined strategy to generate the epigenetic and light chain immunoglobulin (ELCLV3-21) signature to classify 219 individuals with MBL. The ELCLV3-21 high-risk signature distinguished MBL individuals with a high probability of progression (39.9% and 71.1% at 5 and 10 years, respectively). ELCLV3-21 improved the accuracy of predicting time to therapy for individuals with MBL compared with other established prognostic indicators, including the CLL international prognostic index (c-statistic, 0.767 vs 0.668, respectively). Comparing ELCLV3-21 risk groups in MBL vs a cohort of 226 patients with CLL revealed ELCLV3-21 high-risk individuals with MBL had significantly shorter time to therapy (P = .003) and reduced overall survival (P = .03) compared with ELCLV3-21 low-risk individuals with CLL. These results highlight the power of the ELCLV3-21 approach to identify individuals with a higher likelihood of adverse clinical outcome and may provide a more accurate approach to classify individuals with small B-cell clones.

Monoclonal B cell lymphocytosis (MBL) has been shown to be the precursor condition that precedes overt diagnosis of chronic lymphocytic leukemia (CLL). Whereas CLL is classified with greater than or equal to 5 × 109/L B lymphocytes in the peripheral blood, MBL is a clonal expansion of B-cells below this threshold. MBL can be further divided into high- or low-count based on whether the B-cell count is above or below 0.5 × 109/L. Approximately 10% of the population over 40 is estimated to have MBL, increasing to >50% over the age of 90. While low-count MBL is unlikely to progress, 1-2% of high-count (HC) MBL individuals progress to CLL requiring therapy per year. It is debatable if all patients with detectable MBL should be classified as an entity requiring monitoring by a hematologist, especially for low-count MBL. In addition, the diagnosis of leukemia is distressing to patients; therefore, it is important to identify HC MBL patients that are more likely to progress to disease requiring treatment and thus should be monitored more closely. As the majority of MBL cases phenotypically resemble CLL, established prognostic markers including recurrent chromosomal aberrations, beta-2 microglobulin levels, and the mutational status of the Immunoglobulin heavy-chain variable region locus (IGHV) have been shown to predict time to treatment (TTT) and overall survival (OS) in a large retrospective study of MBL1. CLL patients can also be divided into three distinct epigenetic subtypes that reflect progressive DNA methylation changes that occurs during B cell development. These 'epitypes' termed low-programmed (LP), intermediate-programmed (IP), and high-programmed (HP) independently predict clinical outcomes irrespective of disease stage and treatment2. LP-CLL patients follow a generally unfavorable clinical course compared to the more indolent HP-CLL patients, while IP CLL patients display an intermediate outcome. Here we sought to determine if epitype forecasts progression to CLL and eventual clinical outcome for individuals with MBL. We analyzed 66 individuals diagnosed with HC MBL at the Mayo Clinic with a median follow-up of 6.3 years. Developmental epitype was determined using our novel Methylation-iPLEX technique that interrogates 34 CpGs and assigns epitype using a random forest model2. Seventy-seven percent of the MBL cases were assigned to one of the three epitypes: 42.4% HP, 19.7% IP, and 15.2% LP. The residual 23% remained unclassified due to ambiguous (low confidence) epigenetic patterns or insufficient purity (Figure 1A). The overall proportion of HP and IP epitypes in MBL were significantly greater than proportions observed in CLL cohorts (P<0.01). Epitypes remained stable over time as 20/21 cases for which we obtained a high-confidence epitype classification at multiple time points remained unchanged. Epitype significantly predicted MBL individuals progressing to treatment (P=0.001) with LP individuals progressing in a median of 5.6 years (P=0.049 and P=0.0002 versus IP and HP, respectively); median was not reached in HP or IP (Figure 1B). Epitype also predicted overall survival (OS) in MBL (P=0.04), with LP individuals having a significantly shorter OS (median of 11 years versus not reached in IP and HP; P=0.056 and P=0.048 versus IP and HP, respectively). In this study we evaluated a cohort of 66 HC MBL cases and determined that classification using developmental DNA methylation epitypes can be employed in HC MBL to aid in risk stratification. HC MBL patients classified as LP are more likely to progress to requiring treatment and have a significantly reduced OS. The epigenetic classification may help clinicians decide how closely and frequently a HC MBL individual needs to be monitored. Figure 1: (A) Breakdown of the epigenetic subtype assigned to 66 HC MBL samples. (B) Kaplan-Meier analysis of time to treatment and (C) overall survival of MBL patients separated by epitype. 1. Parikh, S. A. et al. Outcomes of a large cohort of individuals with clinically ascertained high-count monoclonal B-cell lymphocytosis. Haematologica103, e237-e240 (2018). 2. Giacopelli, B. et al. Developmental subtypes assessed by DNA methylation-iPLEX forecast the natural history of chronic lymphocytic leukemia. Blood blood.2019000490 (2019). doi:10.1182/blood.2019000490 Disclosures Byrd: Ohio State University: Patents & Royalties: OSU-2S; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding. Shanafelt:Patent: Patents & Royalties: US14/292,075 on green tea extract epigallocatechin gallate in combination with chemotherapy for chronic lymphocytic leukemia; Merck: Research Funding; Polyphenon E International: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; Hospira: Research Funding; Glaxo-SmithKline: Research Funding; Abbvie: Research Funding; Cephalon: Research Funding; Celgene: Research Funding. Parikh:Genentech: Honoraria; Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding. Kay:Agios: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board.

Abstract The characteristic clinical heterogeneity observed in chronic lymphocytic leukemia (CLL) reflects its biological diversity and as a consequence, better understanding of the biology of this disease has led to the identification of a number of useful prognostic tools. Advances in massively parallel sequencing technologies have unearthed genetic and epigenetic heterogeneity and reinforced the concept that specific genetic lesions and the development of genomic complexity can modulate the pathology of CLL. In addition, DNA methylation profiling has shown that CLL patients can be grouped into at least three clinically relevant epigenetic subgroups with distinct outcomes. However, despite this explosion in knowledge, defining the prognosis of individual CLL patients remains a significant clinical challenge. Consequently, there is a need to identify prognostic tests that can provide reliable personalized risk assessments. We have recently shown that a subset of CLL patients manifest telomere dysfunction, which drives genomic instability and clonal evolution. We went on to show an association between short, dysfunctional telomeres and 'high-risk' genetic lesions1,2. Importantly, longitudinal analysis confirmed that tumor cell telomere length remained remarkably stable throughout the course of the disease suggesting that the telomere length is fixed at an early point in disease pathogenesis making it a reliable predictor of both clinical progression and overall survival (OS) regardless of when the measurement is made. In this study, we generated telomere length profiles in 224 newly diagnosed, early stage CLL patients seen at the Mayo clinic in Rochester using high-throughput single telomere length analysis (HT-STELA). We then tested the ability of telomere length to predict time to treatment (TTT) and OS and compared its predictive power with DNA methylation profiling and CLL-IPI scores. Patients with a mean telomere length inside the fusogenic range (IFR) had a significantly shorter time to treatment (P<0.0001; HR = 3.2) and OS (P = 0.0027; HR = 2.3) when compared to patients with a mean telomere length outside the fusogenic range (OFR) (Figure 1A and B). When considering epigenetic subgroups, we found a strong association between telomere length and 'epitype'; high programmed (HP) CLL cells had significantly longer telomeres than intermediate programmed (IP), and low programmed (LP) CLL cells (P = 0.0002 and P<0.0001 respectively). Furthermore, bifurcation of the HP and IP groups into telomere length OFR and IFR subsets identified patients with distinctly different TTT (P = 0.0008; HR = 2.74) and OS (P = 0.004; HR = 2.4). 79/81 (97.5%) of the LP group had short telomere length IFR, but when we divided the LP group above and below the mean telomere length of the fusogenic range (≤/>2.17kb), patients with the shortest telomeres had a significantly worse OS (P = 0.034; HR = 2.83). Telomere length was also strongly associated with CLL-IPI score; the CLL-IPI low-risk group (CLL-IPI 1) had the longest telomeres and the CLL-IPI high-risk group (CLL-IPI 4) had the shortest telomeres (P<0.0001). More heterogeneity in telomere length was observed in the CLL-IPI 1 and 2 groups and patients with telomere length IFR in these 'low-risk' groups showed significantly shorter TTT (P<0.0001; HR = 4.24) and overall survival (P<0.0001; HR = 10.1) (Figure 1C and 1D). Finally, multivariate modelling, using Cox proportional hazards with forward selection, revealed that telomere length retained independent prognostic significance when CLL-IPI score was entered in the model for time to treatment (HR = 1.42; P = 0.023) and OS (HR = 1.68; P = 0.008). In conclusion, telomere length analysis provides more accurate prognostic information about the progression and outcome of CLL patients and can augment the prognostic power of the CLL-IPI for early stage CLL. References Lin TT et al. Blood. 2010;116(11):1899-907. Britt-Compton et al. Leukemia. 2012; 26(4):826-30. Disclosures Norris: Cardiff University: Patents & Royalties: Telomere measurement patent. Shanafelt:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Pharmacyclics: Research Funding; Jansen: Research Funding. Kay:Acerta: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees. Fegan:Napp: Honoraria; Gilead Sciences, Inc.: Honoraria; Roche: Honoraria; Abbvie: Honoraria; Janssen: Honoraria. Baird:Cardiff University: Patents & Royalties: Telomere measurement patents. Pepper:Cardiff University: Patents & Royalties: Telomere measurement patents.

Abstract In chronic lymphocytic leukemia (CLL), it is recognized that germinal center experience by the cell-of-origin has an influence on the malignant phenotype and impacts clinical behavior. Measurement of the mutational status of the IGHV locus is a commonly used approach that subgroups patients according to this natural history. Other biomarkers correlating with these subgroups, such as ZAP70 expression and ZAP70 DNA methylation, are also used. Recently, we and others have found vast differences in global DNA methylation profiles among CLL cases that classifies distinct subgroups related to their natural history. Here we systematically evaluate various markers of natural history of the cell-of-origin for their ability to discriminate clinical outcomes. IGHV mutational status and ZAP70 expression are currently the most widely used prognostic markers of germinal center experience. These markers are generally concordant, with IGHV-unmutated associating with ZAP70 positivity. To compare their relative prognostic impact, we selected CLL cases from a large sample cohort collected as a prospective study maintained by the CLL Research Consortium that were discordant (IGHV-mutated and ZAP70(+) or IGHV-unmutated and ZAP70(-); n=192). We found a significant difference in time-to-first-treatment (TTFT; P=0.002) and overall survival (OS; P=0.032) between discordant groups, with curves separating according to IGHV as opposed to ZAP70 (Fig.1A). DNA methylation-based classification of CLL according to their natural history has not been compared to IGHV status. This classification strategy subgroups the majority of cases into low-programmed CLL (LP-CLL) or high-programmed (HP-CLL) subgroups that generally correlate with IGHV-unmutated/ZAP70(+) and IGHV-mutated/ZAP70(-), respectively, along with a minority of cases in an intermediate (IP-CLL) subgroup. We used our previously-described DNA methylation assay, based on MassARRAY interrogating 7 genomic loci including ZAP70, to group the 192 samples with discordant IGHV/ZAP70 results with 327 samples analyzed previously from the same cohort. DNA methylation (LP-CLL vs. HP-CLL) outperformed IGHV classification (unmutated vs. mutated) in terms of separating TTFT curves (n=432; LP-CLL vs. HP-CLL median difference=4.8 years, unmutated vs. mutated IGHV median difference=2.9 years) and OS curves (n=491; LP-CLL vs. HP-CLL median difference=12.9 years, unmutated vs. mutated IGHV median difference=6.8 years). To evaluate whether DNA methylation subgroups provide additional prognostic information to IGHV, we compared TTFT and OS within IGHV subtypes. We found that the ability of DNA methylation subgrouping to separate OS curves was maintained in the IGHV-unmutated subtype (P<0.001; Fig.1B) and TTFT in both IGHV-unmutated and mutated subtypes (P<0.001 and P=0.016, respectively). Conversely, IGHV subtypes did not significantly separate outcomes within DNA methylation subgroups (P>0.5 for all). Further investigation of the features of DNA methylation subgroups as defined revealed a strong bias in the frequency of several recurrent adverse prognostic somatic mutations. In particular 17p deletions and EGR2, SF3B1 and XPO1 mutations occurred frequently within the LP-CLL subgroup. We next explored whether the presence of each mutation correlated with clinical outcome within its corresponding DNA methylation subgroup, and found heterogenous results. EGR2 mutations separated TTFT in the LP-CLL subgroup (P=0.0009), but 17p, XPO1 and SF3B1 did not (P>0.3). This suggests that the cell-of-origin should be taken into account to accurately assess the clinical impact of somatic mutations. In summary, we demonstrate that among discordant cases for IGHV/ZAP70, IGHV appears to be a stronger prognostic factor for both TTFT and OS. When then evaluating the relative importance of DNA methylation subgrouping and IGHV status on TTFT and OS, we find that DNA methylation subgrouping is the more powerful prognostic factor. Among the markers of the cell-of-origin, DNA methylation is a strong tool for risk-stratifying patients and should be considered in development of risk prediction models. Figure 1. A) Kaplan-Meier analysis of TTFT of IGHV-mutated and ZAP70(+) versus IGHV-unmutated and ZAP70(-) (P=0.002, median difference=4.4 years). B) Kaplan-Meier analysis of OS in patients with unmutated IGHV separated by DNA methylation subgroup (P<0.001). Figure 1. A) Kaplan-Meier analysis of TTFT of IGHV-mutated and ZAP70(+) versus IGHV-unmutated and ZAP70(-) (P=0.002, median difference=4.4 years). B) Kaplan-Meier analysis of OS in patients with unmutated IGHV separated by DNA methylation subgroup (P<0.001). Disclosures Wierda: Acerta: Research Funding; Novartis: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Abbvie: Research Funding. Brown:Infinity: Consultancy; Janssen: Consultancy; Gilead Sciences: Consultancy; Roche/Genentech: Consultancy; Celgene: Consultancy; Sun BioPharma: Consultancy; Pfizer: Consultancy; Abbvie: Consultancy. Kipps:Roche: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding.

AbstractAlterations in global DNA methylation patterns are a major hallmark of cancer and represent attractive biomarkers for personalized risk stratification. Chronic lymphocytic leukemia (CLL) risk stratification studies typically focus on time to first treatment (TTFT), time to progression (TTP) after treatment, and overall survival (OS). Whereas TTFT risk stratification remains similar over time, TTP and OS have changed dramatically with the introduction of targeted therapies, such as the Bruton tyrosine kinase inhibitor ibrutinib. We have shown that genome-wide DNA methylation patterns in CLL are strongly associated with phenotypic differentiation and patient outcomes. Here, we developed a novel assay, termed methylation-iPLEX (Me-iPLEX), for high-throughput quantification of targeted panels of single cytosine guanine dinucleotides from multiple independent loci. Me-iPLEX was used to classify CLL samples into 1 of 3 known epigenetic subtypes (epitypes). We examined the impact of epitype in 1286 CLL patients from 4 independent cohorts representing a comprehensive view of CLL disease course and therapies. We found that epitype significantly predicted TTFT and OS among newly diagnosed CLL patients. Additionally, epitype predicted TTP and OS with 2 common CLL therapies: chemoimmunotherapy and ibrutinib. Epitype retained significance after stratifying by biologically related biomarkers, immunoglobulin heavy chain mutational status, and ZAP70 expression, as well as other common prognostic markers. Furthermore, among several biological traits enriched between epitypes, we found highly biased immunogenetic features, including IGLV3-21 usage in the poorly characterized intermediate-programmed CLL epitype. In summary, Me-iPLEX is an elegant method to assess epigenetic signatures, including robust classification of CLL epitypes that independently stratify patient risk at diagnosis and time of treatment.

Key Points KMT2C mutations occur in 15% and 25% of patients with cHCL and vHCL, respectively, along with CCND3 and U2AF1 mutations each in 13% of vHCLs. NF1, NF2, N/KRAS, and IRS1 alterations contribute to clinical resistance to vemurafenib treatment in patients with cHCL.

Significance In this work we report on the previously uncharacterized clinical and biological role for EGFL7 in acute myeloid leukemia (AML). Patients with increased EGFL7 mRNA expression had lower complete remission rates and shorter overall and event-free survival, demonstrating the clinical relevance of EGFL7 expression in cytogenetically normal AML. Our results show that AML blasts are able to synthesize and secrete EGFL7 protein, promoting autocrine blast cell growth. Inhibition of EGFL7 results in decreased proliferation and induces apoptosis of AML cells. Taken together, our data provide the rationale for targeting EGFL7 using blocking antibodies as a therapy for patients with AML.

Abstract Monosomy of chromosome 7 is the most frequent autosomal monosomy in acute myeloid leukemia (AML), where it associates with poor clinical outcomes. However, molecular features associated with this sole monosomy subtype (-7 AML), which may give insights into the basis for its poor prognosis, have not been characterized. In this study, we analyzed 36 cases of -7 AML for mutations in 81 leukemia/cancer-associated genes using a customized targeted next-generation sequencing panel (Miseq). Global gene and miRNA expression profiles were also determined using paired RNA and small RNA sequencing data. Notably, gene mutations were detected in all the major AML-associated functional groups, which include activated signaling, chromatin remodeling, cohesin complex, methylation, NPM1, spliceosome, transcription factors, and tumor suppressors. Gene mutations in the chromatin remodeling groups were relatively more frequent in patients &lt;60 years of age, who also had less mutations in the methylation and spliceosome groups compared with patients ≥60 years of age. Novel recurrent mutational events in AML were identified in the SMARCA2 gene. In patients ≥60 years of age, the presence of spliceosome mutations associated with a lower complete remission rate (P = 0.03). RNA sequencing revealed distinct gene and miRNA expression patterns between the sole -7 and non -7 AML cases, with reduced expression, as expected, of many genes and miRNAs mapped to chromosome 7, and overexpression of ID1, MECOM, and PTPRM, among others. Overall, our findings illuminate a number of molecular features of the underlying aggressive pathobiology in -7 AML patients. Cancer Res; 77(1); 207–18. ©2016 AACR.