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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 BACKGROUND: The study of rare diseases is limited by the uncommon nature of the conditions as well as the widely dispersed patient populations. Current rare disease registries such as the National Organization of Rare Diseases utilize centralized platforms for data collection; however because of their broad nature, these do not always capture unique, disease specific elements. Hairy Cell Leukemia (HCL) is a rare leukemia globally with approximately 900 new cases diagnosed in the US each year. The HCL Foundation undertook creation of a Patient Data Registry that collects data from multiple HCL Centers of Excellence (COE) around the globe to better understand the complications, treatment outcomes, disease subtypes, comorbid conditions, epidemiology, and quality of life of patients with HCL. METHODS: Investigators at The Ohio State University Department of Biomedical Informatics and Division of Hematology in collaboration with the HCL Foundation developed a Patient Data Registry (PDR) for the longitudinal capture of high quality research data. This system differs from other registries in that it uses a federated( rather than centralized) architecture, wherein data is queried and integrated in an on-demand manner from local registry databases at each participating site. Further, the data collected for use in the registry combines both automated exports from existing electronic health records (EHRs) as well as additional data entered via a set of web-based forms. All manually entered data comes from source documents, and data provenance spanning electronic and manually entered data is maintained via multiple technical measures. Patients may be enrolled at HCL COE, or, if they do not have access to a COE they may enroll via a web-based portal (www.hairycellleukemia.org). At this time due to regulatory requirements the web-based portal is available to US patients only. All data are de-identified (see Figure 1: De-Identification Workflow) which reduces regulatory burden and increases opportunities for data access and re-use. End users have access to data via a project-specific query portal. RESULTS: The Patient Data Registry has been deployed at The Ohio State University, Royal Marsden Hospital, and MD Anderson Cancer Center, and is undergoing deployment at the University of Rochester. Up to 25 international HCL COE may participate. In addition, US patients are actively entering the registry via the web-based portal. To date, 227 patients have been consented to the registry with 119 of these being via the web-based entry point. CONCLUSION: We created an international and web-based patient data registry which will enable researchers to study outcomes in HCL in ways not previously possible given the rarity of the disease. This work was made possible by research funding from the Hairy Cell Leukemia Foundation. Figure De-Identification Workflow Figure. De-Identification Workflow Disclosures Andritsos: Hairy Cell Leukemia Foundation: Research Funding. Anghelina:Hairy Cell Leukemia Foundation: Research Funding. Lele:Hairy Cell Leukemia Foundation: Research Funding. Burger:Pharmacyclics: Research Funding. Delgado:Gilead: Consultancy, Honoraria; Novartis/GSK: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Infinity: Research Funding. Jones:AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding. Lozanski:Beckman Coulter: Research Funding; Genentech: Research Funding; Stemline Therapeutics Inc.: Research Funding; Boehringer Ingelheim: Research Funding. Montserrat:Morphosys: Other: Expert Testimony; Vivia Biotech: Equity Ownership; Gilead: Consultancy, Other: Expert Testimony; Pharmacyclics: Consultancy; Janssen: Honoraria, Other: travel, accommodations, expenses. Parikh:Pharmacyclics: Honoraria, Research Funding. Park:Genentech/Roche: Research Funding; Amgen: Consultancy; Juno Therapeutics: Consultancy, Research Funding. Robak:Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding. Tam:janssen: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees. Heckler:Hairy Cell Leukemia Foundation: Research Funding. Payne:Hairy Cell Leukemia Foundation: Research Funding.

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.

Abstract The majority of pts with newly diagnosed CLL are classified with Rai stage 0 or 1 disease and typically experience an indolent clinical course. However, little is known regarding the reasons for initiating therapy in this population or patterns of initial treatment in the contemporary era of our practicing community. We utilized data from the Connect CLL registry, a US-based multi-center, prospective observational cohort study that is aimed at understanding patterns of CLL management without a study-specific intervention. Eligible pts were adults with a clinical diagnosis of CLL who initiated therapy ≤2 months prior to enrollment, and were enrolled from academic (n=155), community (n=1311), or government (n=28) sites between 2010-2014. Descriptive statistics were applied to examine data on demographics, baseline characteristics, treatment selection, early treatment outcomes, and reasons for initiating therapy that were reported for pts with Rai 0/1 CLL receiving first-line therapy. Multivariable logistic regression models were constructed to evaluate predictors of complete response (CR) and event-free survival (EFS) defined as the occurrence of the first of disease progression, relapse or death within 24 months of enrollment. Among 684 pts who received first-line therapy at enrollment and for whom Rai stage was available, 363 had Rai 0/1 CLL. This group had a median age of 68 years (range 41-90); 94% were white, 60% male, 98% insured, and 93% were treated in community practices; 47% had Rai stage 0 disease (54% Binet A); 33% had ≥1 constitutional symptoms; 96% had ECOG performance status of 0 or 1; 35% had conventional cytogenetics and 53% had FISH analysis performed. Among pts for whom cytogenetic or FISH analysis were performed, 159 (72%) were abnormal, of them 37 pts (23%) had del(11q), 104 pts (65%) had del(13q), 33 pts (21%) had trisomy 12, and 20 pts (13%) had del(17p) by conventional cytogenetics and/or FISH, including 8% with del(17p) by FISH. The median time from diagnosis to initiation of therapy was 2.5 years (range 0-20 years) for Rai 0/1 pts compared with 0.5 years for pts enrolled with Rai stage ≥2 (range 0-32). The most common reasons for initiating therapy (with >1 reason possible) in Rai 0/1 CLL were: bulky or progressive or lymphadenopathy (40%), progressive lymphocytosis (39%), any disease-related symptom (weight loss, fatigue, fever, night sweats; 28%), anemia (20%), and thrombocytopenia (15%); 42% of pts had >1 reason for initiation. The most common first-line regimens used in this cohort were: fludarabine/cyclophosphamide/rituximab (FCR; 29%), bendamustine/rituximab (BR; 20%), and rituximab alone (12%). Of the 317 pts who either responded to treatment during the first 24 months of enrollment or were followed ≥24 months, 143 pts (45%) achieved CR (investigator assessed as per protocol) and 264 (83%) achieved CR or partial response. Female gender (odds ratio [OR] = 2.22), treatment with BR or FCR (OR = 3.41), and time from diagnosis to therapy (OR = 0.92; with each year increase in time from diagnosis to initiation of therapy, relative odds of CR decreases by 8%), were independent predictors of CR. Of the 311 evaluable pts, 82 pts (26%) experienced an EFS event during the 24 months on study, of which 19 (23%) were deaths and 48 (59%) were progressions. Age ≥75 years (OR = 4.46), lymphocytosis as a reason for therapy initiation (OR = 1.89), and male gender (OR = 1.73) were independent predictors of an EFS event within 24 months. The Connect CLL registry represents the largest comprehensive prospective cohort of CLL pts collected from multiple practices across the US. In concordance with other studies, pts with Rai 0/1 initiated therapy approximately 2.5 years following CLL diagnosis and included a cohort of pts with poor prognostic FISH characteristics. Although chemoimmunotherapy was associated with a significantly higher likelihood of CR, progressive lymphocytosis as a reason for initiating therapy (and not any form of treatment) was more likely to predict an early EFS event. Approaches using novel agents or combination therapies aimed towards addressing pts with progressive lymphocytosis should be considered in future studies. Disclosures Flowers: Seattle Genetics: Consultancy; OptumRx: Consultancy; AbbVie: Research Funding; Pharmacyclics: Research Funding; Gilead Sciences: Research Funding; Genentech: Research Funding; AbbVie: Research Funding; Celegene: Other: Unpaid consultant, Research Funding; Spectrum: Research Funding; Onyx Pharmaceuticals: Research Funding; Spectrum: Research Funding; Acerta: Research Funding; Gilead Sciences: Research Funding; Janssen: Research Funding; Infinity Pharmaceuticals: Research Funding; Infinity Pharmaceuticals: Research Funding; Janssen: Research Funding; Millennium/Takeda: Research Funding; Onyx Pharmaceuticals: Research Funding; Pharmacyclics: Research Funding; Acerta: Research Funding; Millennium/Takeda: Research Funding. Nabhan:Celgene Corporation: Honoraria, Research Funding. Kay:Pharmacyclics: Research Funding; Genentech: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hospira: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharma: Research Funding. Mato:AbbVie: Consultancy, Research Funding; Janssen: Consultancy; Celgene Corporation: Consultancy, Research Funding; Pronai Pharmaceuticals: Research Funding; Pharmacyclics: Consultancy, Research Funding; TG Therapeutics: Research Funding; Gilead: Consultancy, Research Funding; Genentech: Consultancy. Lamanna:Genentech-Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Infinity: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Grinblatt:Celgene Corporation: Consultancy, Honoraria, Speakers Bureau. Kipps:Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor; Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor. Kozloff:Roche: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Genentech: Consultancy, Speakers Bureau; AbbVie: Consultancy. Sullivan:Celgene Corporation: Employment, Equity Ownership. Flick:Celgene Corporation: Employment, Equity Ownership. Kiselev:Celgene Corporation: Consultancy. Bhushan:Celgene Corporation: Employment, Equity Ownership. Swern:Celgene Corporation: Employment, Equity Ownership. Sharman:TG Therapeutics, Inc.: Research Funding; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Research Funding; Calistoga: Honoraria; Celgene Corporation: Consultancy, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Roche: 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.

Abstract Abstract 1614 Poster Board I-640 Background Cytogenetic abnormalities in B-cell CLL are important prognostic indicators for predicting disease progression and treatment response. Until recently, only interphase cytogenetics has been readily applicable to clinical use in CLL due to the inability of traditional mitogens to promote effective metaphase cytogenetic identification. Recently, cytokine or CpG oligonucleotide stimulation has been utilized to promote efficient metaphase analysis. One concern with this approach is the actual clonality and reproducibility of such clones when examined by different cytogenetic laboratories. The CLL Research Consortium (CRC) conducted a prospective trial to test (1) whether CpG would enhance detection of abnormal clones and (2) whether the CRC cytogenetic laboratories would achieve similar results when testing the same patient samples. Methods Initially, one laboratory compared stimulation of CLL cells using CpG versus pokeweed mitogen (PWM) + 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in the same samples. To test the reproducibility of CpG stimulation, fresh blood samples from 1 normal control and 12 CLL patients were collected at one site and distributed (blinded to phenotype and prior cytogenetic results) to 5 CRC cytogenetic laboratories where they were cultured for 3 days using CpG stimulation, harvested, banded and analyzed utilizing standard laboratory procedures. When possible, each laboratory analyzed 20 metaphases per case. Interphase FISH analysis for CLL (including probes for chromosomes 6q, 11q, 12, 13q, 17p, and IGH/CCND1) was also done on each sample. Results In the PWM+TPA versus CpG comparison, more cases were found to have clonal abnormalities with CpG stimulation (147 of 229, 64%) than with PWM+TPA (110 of 229, 48%; P=0.0005). All clonal abnormalities detected in the PWM+TPA cultures were also observed in the CpG cultures. In the 5-laboratory CpG comparison, the results were compiled and compared to the concurrent FISH results. The CpG karyotypes were concordant with the FISH results. The control case had a normal CpG-stimulated karyotype in all 5 laboratories. Three cases exhibited a complex karyotype; one of these also exhibited 11q-, one had 17p-, and one had neither. The cytogenetic descriptions varied modestly among labs, but all identified the complex clones. Every case including the control exhibited some nonclonal abnormal cells, but no pattern was detected among the 5 labs. When an abnormal clone was found by only 1 or 2 labs, it was present in only a very small number of cells. A small 13q- clone was observed in 2 cases in 1 and 2 labs (3 of 20 and 5 of 50 cells, respectively), and was confirmed by interphase FISH. A single lab found 2 of 30 cells with a t(12;15) in one case. Conclusions Abnormal clones in CLL are more readily detected with CpG stimulation than with traditional B-cell mitogens. The clonal abnormalities revealed by CpG stimulation are consistent with the “gold standard” interphase FISH results, and are reproducible among different cytogenetic laboratories. We have no evidence that CpG oligonucleotides create new clonal B-cell populations in vitro during 3 days of cell culture. CpG stimulation in vitro reveals clonal cytogenetic abnormalities and complexity in CLL that should be evaluated as potential independent prognostic parameters. Disclosures Kay: Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees; Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding.

Precis: This study is the first to report micropulse transscleral cyclophotocoagulation (MP-TSCPC) use in only good vision patients. MP-TSCPC significantly reduced intraocular pressure (IOP) and glaucoma medication use without any significant reduction in visual acuity at every postoperative follow-up point. Purpose: To evaluate outcomes of MP-TSCPC in eyes with baseline best-corrected visual acuity (BCVA) of ≥20/60. Methods: A retrospective review of patients who underwent MP-TSCPC at Mayo Clinic and Ross Eye Institute from July 2016 to August 2017 with BCVA of ≥20/60, and a minimum of 3 months follow-up. Results: A total of 61 eyes of 46 patients (68.80±17.12 y) underwent MP-TSCPC with a mean follow-up of 10.2±3.1 months. Mean IOP and mean number of glaucoma medications used were significantly reduced from baseline at every follow-up time point ( P <0.0001). At month 12, mean IOP was reduced 40.2% from baseline with 85.4% of the patients having an IOP reduction of ≥20%, and mean glaucoma medication use reduced by 0.82±0.53 with 79.6% of the patients having a reduction of ≥1 medication. There was no significant reduction in BCVA from baseline at any follow-up point ( P >0.05), except for 10 eyes with a vision loss of ≥2 lines and 5 out of 10 eyes had cataract progression. The probability of complete success (IOP range, 6 to 21 mm Hg or ≥20% IOP reduction; BCVA loss ≤2 lines, no reoperation for glaucoma) was 74.14%, 83.61%, 84.21%, and 75.0% at months 1, 3, 6, 12, respectively. The probability of qualified success (above criteria for IOP, no reoperation and BCVA loss >2 lines) was 81.03%, 91.80%, 94.74%, and 93.75% at months 1, 3, 6, 12, respectively. Conclusions: MP-TSCPC should be considered earlier in the management of glaucoma and can possibly be offered as an alternative to incisional glaucoma surgeries.

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.

Significance Glioblastoma, the most common and lethal of primary brain tumors, can diffusely infiltrate brain. Because the components of the cytoskeleton that drive tumor invasion are nonredundant, they have been attractive targets for blocking glioblastoma dispersion. However, we find that while targeting one of these—the molecular motor myosin IIA—effectively blocks glioblastoma invasion, it enhances tumor proliferation and lethality. In this study, we have found that myosin IIA functions as a tumor suppressor by reducing signaling of two oncogenes—ERK1/2 and NF-κB—in a manner regulated by the mechanics of the tumor and surrounding brain. Our results also argue that effective treatment of glioblastoma will require inhibiting targets that drive both invasion and proliferation.

AbstractContextInactivating germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene are linked to pituitary adenoma predisposition. Here, we present the youngest known patient with AIP-related pituitary adenoma.Case DescriptionThe patient presented at the age of 4 years with pituitary apoplexy and left ptosis with severe visual loss following a 1-year history of abdominal pain, headaches, and rapid growth. His IGF-1 level was 5× the upper limit of normal, and his random GH level was 1200 ng/mL. MRI showed a 43 × 24 × 35‒mm adenoma with suprasellar extension invading the left cavernous sinus (Knosp grade 4). After transsphenoidal surgery, histology showed a grade 2A sparsely granulated somatotropinoma with negative O6-methylguanine-DNA methyltransferase and positive vascular endothelial growth factor staining. Genetic testing identified a heterozygous germline nonsense AIP mutation (p.Arg81Ter). Exome sequencing of the tumor revealed that it had lost the entire maternal chromosome-11, rendering it hemizygous for chromosome-11 and therefore lacking functional copies of AIP in the tumor. He was started on octreotide, but because the tumor rapidly regrew and IGF-1 levels were unchanged, temozolomide was initiated, and intensity-modulated radiotherapy was administered 5 months after surgery. Two months later, bevacizumab was added, resulting in excellent tumor response. Although these treatments stabilized tumor growth over 4 years, IGF-1 was normalized only after pegvisomant treatment, although access to this medication was intermittent. At 3.5 years of follow-up, gamma knife treatment was administered, and pegvisomant dose increase was indicated.ConclusionMultimodal treatment with surgery, long-acting octreotide, radiotherapy, temozolomide, bevacizumab, and pegvisomant can control genetically driven, aggressive, childhood-onset somatotropinomas.