IgVH Mutation Research Articles

NK Cell Dysfunction in CLL Is Mediated through SHP-1 Signaling and Is Associated with Poor Prognosis

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, presenting with a highly variable clinical course, outcome and response to therapy. This variability has been linked to a complex tumor microenvironment and genetic/epigenetic modifications that lead to immunosuppression. Indeed, the clinical hallmark of this disease is the susceptibility to infections and secondary malignancies. While T cell dysfunction in CLL has been well described, conflicting information exists on NK cell changes in CLL and their role in CLL immunosurveillance. Here, we analyzed samples from 75 untreated (or completed treatment >2 years ago) CLL patients and 30 healthy controls. We discovered a substantial variation in NK cell function compared with healthy controls, which we further classified into hyperfunctional, normofunctional and dysfunctional based on their ability to degranulate, and produce IFN- γ in response to K562 targets ( Figure 1). NK cells from ‘dysfunctional’ CLL samples also displayed impaired cytotoxicity against K562 targets, weaker synapse formation and Syk/Zap70 signaling in response to antibody-dependent cellular cytotoxicity (ADCC) compared to those from ‘functional’ samples. The NK functional status was strongly associated with multiple prognostic markers, including cytogenetics, IgVH mutation status, ZAP-70 expression and disease stage, with patients with dysfunctional NK cells having significantly higher rates of secondary malignancies and viral infections ( Table 1). Using multispectral flow cytometry we discovered high phenotypic heterogeneity with unique NK cell clusters present in the dysfunctional samples. Those clusters express a pattern of activation-induced exhaustion, with upregulation of multiple checkpoint molecules such as PD-1, TIGIT, LAG-3, KLRG-1, TIM-3 but not evidence of terminal differentiation (CD57 negative). Those clusters also showed concomitant expression of activating molecules such as NKG2D and DNAM-1, suggesting reversibility of their dysfunctional status. We subsequently found significantly higher (p<0.0001) constitutive phosphorylation of SHP-1 protein, a phosphatase downstream of multiple inhibitory NK cell receptors, in NK cells from dysfunctional samples. Treatment with a SHP-1 inhibitor NSC-87877 (EMD Millipore) or use of siRNA targeting SHP-1 (ThermoFisher scientific), resulted in reversal of NK dysfunction with full restoration of their functional capacity. Taken together, our data suggest that NK cells functional status plays an important role in CLL disease progression and secondary complications. The novel mechanism of NK cell immune dysfunction in CLL described here has the potential to improve outcomes with novel immunotherapies such as chimeric antigen receptors, checkpoint inhibitors and bi-specific antibody therapy that have shown disappointing results in CLL to date when compared with other B cell malignancies.

IDENTIFY THE MOST COMMONLY MUTATED GENES IN CHRONIC LYMPHOCYTIC LEUKEMIA AND THE SIGNIFICANCE ON PROGNOSIS

Introduction: The prognosis of chronic lymphocytic leukemia (CLL) varies widely individually. The clinical staging systems by Rai and Binet, cytogenetics/FISH, and mutational status of IGHV have been commonly used as clinical prognostic factors for CLL patients. The advent of molecular panels by next-generation sequencing might provide additional markers to predict the outcome of CLL patients. In this study, we evaluate gene mutation profiles as well as IGVH mutation status for its application to predicting survival outcomes among these CLL patients. Methods: We performed next-generation sequencing (NGS) sequencing in 196 clinically characterized CLL samples in OSU James Cancer Hospital and analyzed the IGVH mutation status of 172 patients from the same cohort. In addition, clinic-pathological data include age, gender, immunophenotyping, and cytogenetics/FISH. Results: 50 genes in the NGS mutation panel and IGVH mutation status are explored for a sequencing-based prediction model. Five genes (TP53, NOTCH1, SF3B1, and RAS including KRAS and BRAF) are identified as the most commonly mutated genes in the order of frequencies from 24.1%, 14.2%, 13.4%, and 11.6%, respectively. There are 66.9% unmutated IGVH genes but has no significant correlation between the outcomes (p = 0.48). However, the patients with unmutated IGVH have a substantial correlation to mutated TP53 and NOTCH1, but not SF3B1 and RAS mutations. The outcome in this cohort is significantly correlated to the pathogenic mutations present among these four genes (p < 0.001, n = 139, Cochran’s Q test). However, both Kaplan-Meier and Cox Regression survival analyses reveal that mutated RAS (BRAF and KRAS) is the only one that has a significant correlation to a favorable survival outcome. Keywords: chronic lymphocytic leukemia (CLL), genomics, epigenomics, and other -omics, risk models No conflicts of interests pertinent to the abstract.

ODP090 Fragility Fractures and Osteoporosis Screening in Patients with Chronic Lymphocytic Leukemia

Abstract Chronic lymphocytic leukemia (CLL) and osteoporosis are diseases of older adults with high prevalence rates. CLL infiltrates the bone marrow, increasing bone resorption and demineralization which contribute to the pathophysiology of osteoporosis. Despite this, there are limited clinical data describing the association of CLL with osteoporosis and fracture risk. We hypothesized that osteoporosis and fragility fractures (FF) are common in patients with CLL. The Huntsman Cancer Institute (HCI) has a CLL database which includes all patients diagnosed with CLL since January 1, 2000 and received care at the HCI. Patients included were identified from the CLL database with ICD-9 and 10 codes associated with osteopenia, osteoporosis and/or presence of a FF. Of the 57 patients included, 67% were female (n=38) with a mean age of 69 ± 10 years and 93% Caucasian. 60% (n=34) had RAI stage of 0-1 with genetic changes noted as follows: Del(13q) 46% (n=26), Del(17p) 7% (n=4), Del(11q) 5% (n=3), and Trisomy 12 14% (n=8). IGVH mutation was present in 31.6% of patients (n=18). 47% (n=25) of patients were treated with chemotherapy, of which Rituximab (n=14), Bendamustine (n=7), Cyclophosphamide (n=5), and Ibrutinib (n=5) were the most frequently used. 58.5% (n=32) had a history of FF, with 84% occurring in the spine (n=27/32). Assessment for Vitamin D deficiency and hyperparathyroidism were performed in 54% (n=31) and 21% (n=12) of the cohort respectively. Even though every patient in the study either had a diagnosis of osteoporosis by ICD code or FF, only 44% (n=25) of patients had an available DXA scan for our review, and only 31% (n=18) received treatment for osteoporosis. Of those with a DXA, the worst T score was in the right femoral neck (T score -1.9, IQR -1.5 to -2.5) with 44% (n=14/32) either having T scores in the normal or osteopenic range. Interestingly, FF was associated with higher T scores in the spine (FF spine T score -1. 0±1.5 vs no FF T score -2.2±0.8, p=0. 03). Clinical factors associated with FF were male sex (83% men vs 46% women, p&amp;lt;0. 01) and positive smoking history (93% smokers vs 46% non-smokers, p&amp;lt;0. 01). Use of chemotherapy and hip T scores were not associated with FF. The data from our study suggests that patients with CLL are predisposed to axial fragility fractures despite non-osteoporotic T scores. In addition, osteoporosis screening with DXA is under-utilized in the CLL population, despite meeting age-appropriate screening guidelines. Increased awareness and screening for axial fragility fractures, especially in men and smokers, should be recommended in the long term follow up of patients with CLL. Further study is needed to understand the pathophysiology of osteoporosis and FF in patients with CLL to develop improved methods for prevention of these morbid fractures. Presentation: No date and time listed

CLLU1 Expression Is Not Confined to Chronic Lymphocytic Leukemia Cells; Putative Role of Gene-Body Hypomethylation in Regulating Splice Variant-Specific Transcriptional Dynamics

Background: Alternative splicing of the CLLU1 gene generates 6 transcript variants (accession numbers from the National Center for Biotechnology Information: AJ845162-AJ845167), two of which comprise a putative coding DNA sequence (AJ845165 and AJ845166, CDS transcripts). Previous studies showed that CLLU1 overexpression is highly confined to chronic lymphocytic leukemia (CLL) cells and that only the AJ845162 is expressed in both high- and low-risk CLL patients, while expression of the CDS-transcripts is confined to the high-risk subgroup.Pair-wise linear regression analysis revealed a powerful linear relation between the expression levels of all the splice variants, thereby it was deduced that CLLU1 transcription is orchestrated by a common promoter, which is located upstream of CLLU1 exon 1 (Buhl et al., Leukemia 2009). We recently documented (Papamichos et al., Haematologica 2016; s1) that this promoter was evolutionarily derived by an endogenous retrovirus sequence.Herein we argue that CLLU1 expression is not restricted to CLL cells while also we highlight that transcription of CDS transcripts is regulated by an alternative intronic promoter.Methods:CLLU1 high-throughput sequencing of polyadenylated RNA (RNA-Seq) data across 51 human tissues and 2 human cell lines, generated by the Genotype-Tissue Expression (GTEx) project of the US National Institutes of Health Common Fund (GTEx Analysis Release V6p), were analyzed via GTEx Portal. CLLU1 GeneChip expression data (AJ845164 transcript-specific probe set 1558185_at from Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array), generated by Genevestigator project, were downloaded from Genevisible portal.CLLU1 chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-Seq) on histone H3 protein subunit modified at lysine position 4 with trimethylation (H3K4me3) as well as transcription factor (TF) ChIP-Seq data were downloaded from the Encyclopedia of DNA Elements (ENCODE) Regulation supertrack, which is available in UCSC Genome Browser Database. Data on CLLU1 gene-body methylation status in 63 cell lines from ENCODE were downloaded from the HAIB Methyl450 ENCODE track.Results:CLLU1 GeneChip expression data show that the AJ845164 transcript (corresponding to the RP11-693J15.4 long non-coding RNA) is promiscuously expressed in various normal B-cell subpopulations andin mature B-cell neoplasms other than CLL, especially in mantle cell lymphoma.RNA-Seq data analysis reveals that AJ845162 transcript is expressed in normal somatic tissues such as skeletal muscle, salivary gland, and esophageal mucosa. Vice versa, expression of CLLU1 CDS transcripts is confined to transformed lymphocytes.Structure interrogation of CDS transcripts indicates that these variants initiate from an alternative transcriptional start site which is located 2429 nucleotides downstream from the canonical one. H3K4me3 ChIP-Seq data signify the presence of an alternative intronic promoter in this genomic segment. TF Chip-Seq data reveal that the alternative promoter region comprises high affinity binding sites for signal transducer and activator of transcription 3 (STAT3) and Activator protein 1 (AP-1). Analysis of CLLU1 -body methylation status in five lymphoblastoid cell lines reveals that the alternative promoter remains heavily methylated in these cell types. This finding is in accordance with previous studies reporting that CLLU1 gene body is hypermethylated in B-cells from healthy individuals and in IgVH-mutated (IgVH-M) CLL cases while appears hypomethylated in IgVH-unmutated (IgVH-UM) CLL cells.Conclusion: Not all CLLU1 transcripts are equal in discriminating CLL from other mature B-cell malignancies.Whilst of retroviral origin, CLLU1 upstream promoter is not subjected to stringent epigenetic repression but remains active in somatic cells, a tantalizing finding that warrants further investigation.The level of intragenic DNA methylation represents a decisive regulator of alternative promoter usage (Neri et al., Nature 2017). It would be tantalizing to speculate that CLLU1 -body hypomethylation, occuring exclusively in IgVH-unmutated CLL, is mechanistically linked to the epigenetic derepression of the alternative intronic CLLU1 promoter and the subsequent high expression of CLLU1 CDS transcripts in high-risk CLL. DisclosuresNo relevant conflicts of interest to declare.

Real-World Testing and Treatment Patterns in Chronic Lymphocytic Leukemia (CLL) in the United States during the Modern Era: A SEER Patterns of Care Analysis

Introduction: The use of fluorescence in situ hybridization (FISH) and IgVH mutation analyses were initially used to help prognosticate CLL patients in clinical trials. In the modern era of Bruton tyrosine kinase inhibitors and improved response rates in patients with deletion 17p, and demonstrated long-term remissions among mutated IgVH patients treated with fludarabine, cyclophosphamide and rituximab (FCR), the use of testing prior to treatment is now considered standard of care to determine appropriate therapy. Several therapies were FDA approved for CLL since 2008 through 2014, including bendamustine and novel therapeutics (obinutuzumab, ibrutinib, and idelalisib). We aimed to look at patterns of real-world testing frequencies and treatment patterns by comparing two United States population based samples of untreated and treated CLL patients in 2008 and 2014.Methods: The National Cancer Institute sponsored Patterns of Care (POC) study was conducted in a population of CLL patients, randomly sampled from 13 Surveillance, Epidemiology, and End Results (SEER) program registries, diagnosed in 2008 and again in 2014. Trained abstractors collected data on demographics, diagnostics, testing, and treatments from patient medical records within 24 months post-diagnosis (along with a physician verification survey). Patient characteristics were described by age, sex, race, Charlson comorbidity index, area-based socioeconomic status, insurance status, receipt of systemic therapy, and type of treatments by diagnosis year. Patient characteristics and frequency of testing (FISH and IgVH mutation analyses) in 2008 and 2014 were compared using Rao-Scott chi-square tests. Logistic regression taking into account sampling weights was used to estimate odd ratios and 95% confidence intervals for receipt of FISH testing, both unadjusted and adjusted for age, sex, race, insurance status, and receipt of systemic treatment. Adjusted models were run overall and stratified by age at diagnosis (<65 and ≥65).Results: This study included 1,008 CLL patients diagnosed in 2008 and 1,367 CLL patients diagnosed in 2014. The mean age was 69.6 years in 2008, and 68.0 years in 2014. Among those with a known status for FISH testing, the proportion of patients who underwent testing was 51.3% in 2008, which increased to 59.6% in 2014 (p<0.01). Among those with a known status for IgVH mutation analyses, 8.5% underwent testing in 2008, which increased to 15.1% in 2014 (p<0.01). Older age was a strong predictor for not receiving FISH testing in both 2008 and 2014 (adjusted p-trend <0.01 for both years). In 2014, Hispanic patients were less likely to undergo testing (aOR 0.68, 95% CI: 0.48-0.96) compared to non-Hispanic whites, but no significant difference was found among non-Hispanic blacks. Among patients 65 or older, those with Medicare without private insurance were less likely to receive testing compared to Medicare patients with private insurance (aOR 0.49, CI: 0.32-0.74).Most CLL patients were untreated within the first two years from diagnosis (74.3% in 2008, and 70.6% in 2014). FISH testing occurred in a majority but not all patients who received therapy (64.5% of treated patients in 2008, increased to 74.7% in 2014). In 2008, the four most frequently used regimens for first treatment were 1) FCR, 2) rituximab monotherapy, 3) FR, and 4) chlorambucil based regimens. This is in contrast to 2014, where the regimens were 1) bendamustine and rituximab (BR), 2) rituximab monotherapy, 3) FCR, and 4) ibrutinib. When evaluating the use of novel therapies (obinutuzumab, ibrutinib, and idelalisib) for those diagnosed in 2014, there were no significant differences in distribution by age, race/ethnicity, comorbid conditions, SES, or insurance type.Conclusions: There has been an increase in frequency of FISH testing and IgVH mutation analyses from 2008 to 2014. FISH testing is more common compared to IgVH mutation analyses which was modest in both years. Patients of older age, Hispanic ethnicity, and Medicare with no private insurance were also less likely to receive FISH testing in 2014. There are significant differences in the treatment patterns of patients in 2008 compared to 2014, with the use of bendamustine being more common at the expense of FCR. These results highlight rapidly changing practices within CLL in the modern era. DisclosuresNo relevant conflicts of interest to declare.

ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile

AbstractThe presence or absence of somatic mutations in the expressed immunoglobulin heavy chain variable regions (IgVH) of chronic lymphocytic leukemia (CLL) cells provides prognostic information. Patients whose leukemic cells express unmutated IgVH regions (Ig-unmutated CLL) often have progressive disease, whereas patients whose leukemic cells express mutated IgVH regions (Ig-mutated CLL) more often have an indolent disease. Given the difficulty in performing IgVH sequencing in a routine diagnostic laboratory, this prognostic distinction is currently unavailable to most patients. Pilot gene expression profiling studies in patients with CLL identified genes that were differentially expressed between the Ig-unmutated and Ig-mutated CLL subtypes. Here, we have profiled an expanded cohort of 107 patients and show that ZAP-70 is the gene that best distinguishes the CLL subtypes. Ig-unmutated CLL expressed ZAP-70 5.54-fold more highly than Ig-mutated CLL (P < 10-21). ZAP-70 expression correctly predicted IgVH mutation status in 93% of patients. ZAP-70 expression and IgVH mutation status were comparable in their ability to predict time to treatment requirement following diagnosis. In 7 patients, ZAP-70 expression and IgVH mutation status were discordant: 4 Ig-mutated CLLs had high ZAP-70 expression and 3 Ig-unmutated CLLs had low ZAP-70 expression. Among these ZAP-70 “outliers,” those with Ig-mutated CLL had clinical features that are uncharacteristic of this CLL subtype: 2 required early treatment and 2 used a mutated VH3-21 gene, an IgVH gene that has been associated with progressive disease. We developed reverse transcriptase–polymerase chain reaction and immunohistochemical assays for ZAP-70 expression that can be applied clinically and would yield important prognostic information for patients with CLL.

MiR-34a and miR-29b as indicators for prognosis of treatment-free survival of chronic lymphocytic leukemia patients in Chinese Uygur and Han populations

The abnormal expression of miRNAs may play critical roles in the occurrence, development and prognosis of chronic lymphocytic leukemia (CLL), with potential ethnic differences being involved. p53 and immunoglobulin heavy chain variable region gene (IGVH) mutations were monitored and miRNA profile screening of CD19 + cells from Uygur CLL patients was performed, analyzed by miRNA arrays and verified using real-time PCR. There were 68 differentially expressed miRNAs in CD19 + B lymphocytes obtained from 6 Uygur CLL patients, of which miR-1295, miR-29b, miR-34a, miR-21 and miR-29c were the 5 most upregulated, and miR-181a, miR-126, miR-181b, miR-125a-5p and miR199b the 5 most downregulated miRNAs. miR-15a/miR-16-1 which might be important drivers of the disease, were not eliminated by profile screening. From the 68 differentially expressed miRNAs, 5 previously-reported CLL-related miRNAs were selected for further confirmation analyses, from which expression levels of miR-29b, miR-34a and miR-155 were found to be increased while miR-181a and miR-181b decreased. However, there were no differences in the expression levels of miR-15a/miR-16-1 between CLL patients and healthy donors, but the expression levels of miR-15a/miR-16-1 in CLL patients with a 13q deletion was depressed. In addition, there was no difference in the expression level of the above 7 miRNAs between 44 Han and 40 Uygur CLL patients. The expression levels of miR-29b, miR-181a and miR-181b correlated with IGVH mutations, while the expression levels of miR-34a, miR-29b and miR-181b correlated with a p53 abnormality in 84 Uygur and Han CLL patients. Taking p53 abnormality as the cut-off value criteria, low expression levels of miR-34a (cut-off value 4.65, P = 0.02) and miR-29b (cut-off value 4.71, P = 0.009) hinted at a poor treatment-free survival (TFS) prognosis for all CLL patients. Thus miR-34a and miR-29b may represent useful indicators for the prognosis of both Uygur and Han CLL patients.

PB1882 DECONSTRUCTING RICHTER SYNDROME TUMOR NICHE: JAGGED‐1 LIGAND LEADS THE WAY

Background:Richter Syndrome (RS) is defined as the occurrence of a high‐grade lymphoma (usually diffuse large B‐cell lymphoma ‐ DLBCL) in patients with a previous/concomitant diagnosis of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL).RS usually occurs in lymph‐nodes, but sometimes may infiltrate extranodal sites.Isolated cardiac involvement is very unique.Most DLBCL‐type RS does not share lesions with de novo subtypes.NOTCH1 mutation was reported in almost 30% of cases,but NOTCH1 signaling activation independent of its mutational status,as NOTCH ligands mapping in tumor niche, remain scarcely estimated, as opposed to CLL.This has directed our attention to the role of cell non‐autonomous NOTCH1 activation at the cross‐roads between other pathways coming from the niche (i.e. immune checkpoint axis). CLL cells also express the NOTCH ligand JAGGED1 (JAG1), that contributes to CLL pathogenesis by activating its own signaling.Experimental proofs in other settings were made, linking the NOTCH1‐JAG1 signaling to the immunological niche.Strikingly, DLBCL‐type RS exhibits PD‐1/PD‐L1 axis upregulation in up to 80% of cases.Taking advantage of these findings, we decided to perform immunohistochemistry (IHC), using validated antibodies,to dissect the precise anatomy of the NOTCH/ligand interface in RS,in vivo.Aims:1) To investigate context‐dependent, cell‐autonomous/cell non‐autonomous NOTCH1 network activation, using IHC stains. Specifically, we focused on NOTCH1 and its ligand JAG1, a well‐known pivotal player in embryonic heart and cardiac diseases. 2) To zoom into the PD‐1/PDL‐1 axis activation in immune niche, and crosslink the immune‐evasion pathway to tumor oncogenic promotion.Methods:A 69‐year old female was admitted to the Cardiology Intensive Care Unit (ICU), for tachy‐/bradycardia. A cardiac computed tomography identified a voluminous rounded mass at the interatrial septum.The pathologic diagnosis was consistent with non‐GC DLBCL.Bone marrow (BM) biopsy detected CLL/SLL.Molecular analysis did not reveal SF3B1,NOTCH1 and TP53 alterations but evidenced the same IgVH mutation in both mass and BM specimens,proving a clonally‐related RS.Results:IHC stains for the cleaved NOTCH1‐intracellular domain (ICD) protein was negative on both RS lymphoma cells and the surrounding stroma, as in BM specimen. On the contrary, JAG1 was markedly positive both on large neoplastic lymphocytes and in the stromal compartment (Figure 1A). Conversely, JAG1 staining was moderate‐to‐weak on CLL lymphocytes in the BM, as well as in the stromal scaffold (Figure 1B), indicating differential expressions. Unexpectedly, JAG1 upregulation occurred independently of NOTCH1 receptor co‐activation. According to this, JAG1 may stimulate other pro‐survival cascades in the neoplastic B cells, transducing microenvironmental cues, coming from TME, where JAG1 itself was found highly expressed. In addition, we performed IHC stain for PD‐1, proving strong positivity on tumor‐infiltrating lymphocytes (TILs) (Figure 1C); moreover, PD‐L1 marked both neoplastic cells and stromal meshwork in the cardiac TME (Figure 1D).Summary/Conclusion:Our report proved the importance of IHC in characterizing JAG1‐NOTCH1 ligand‐receptor interactions in the RS pathogenesis, in order to anatomically characterize cell‐extrinsic pathways in vivo. We traced JAG1 upregulation, independently of NOTCH1 signaling activation and described PD‐1/PD‐L1 axis functional switching in the context of a very unique extra‐nodal RS localization, thus identifying novel potential actionable therapeutic targets.image

Expression and prognostic relevance of calcium calmodulin-dependent protein kinase kinase 2 (CaMKK2) in chronic lymphocytic leukemia (CLL).

e19002 Background: CaMKK2 has restricted tissue expression in humans, regulates diverse cell functions, and is overexpressed in several human malignancies. Blocking its function reduces cancer cell migration and survival in vitro and in vivo. The relevance of CaMKK2 signaling in lymphoma is uncertain, however. Here, we sought to characterize CaMKK2 expression in CLL, as well as its predictive variables and prognostic relevance. Methods: B cell samples from 39 CLL patients and 4 healthy donors were obtained from a biorepository at our institution through an active protocol. Commercially available kits were used for RNA isolation, cDNA generation, and RT-PCR. The Kolmogorov-Smirnov test was used to compare CaMKK2 expression between patients and donors. Regression analysis was used to identify independent predictors of CaMKK2 expression. Treatment-free survival was estimated using the Kaplan-Meier method. Results: CaMKK2 mRNA was standardized to actin and overexpressed in CLL cells v. healthy donor B cells (2.27 - 11.83 x 10-2 v. 0.93 - 1.67 x 10-2, p = 0.0014). Nine variables were evaluated for possible association, including age, gender, race, WBC and monocyte count, IGVH mutation status, FISH panel, Zap70 and CD38 status. By univariate analysis, unmutated IGVH and African-American race were associated with higher CaMKK2 expression (p = 0.0023 and 0.0238). Race was identified as a potential confounder for IGVH mutation status and vice versa. In a multivariate model, only IGVH mutation status predicted for CaMKK2 expression. Patients in the highest quartile of CaMKK2 expression had lower 2 year treatment-free survival compared with all others (10.7% v. 59.2%, p = 0.0054). Conclusions: CaMKK2 mRNA is over-expressed in CLL cells relative to normal B cells. Higher CaMKK2 expression in CLL may be associated with unmutated IGVH and African-American race and predict shorter time to treatment. Our analyses are limited by all unmutated IGVH patients receiving early treatment in our cohort. [Table: see text]

THE ROLE OF THE GENETIC ABNORMALITIES, EPIGENETIC AND microRNA IN THE PROGNOSIS OF CHRONIC LYMPHOCYTIC LEUKEMIA

Chronic lymphocytic leukemia (CLL) isincreased proliferation ofB-cells with peripheral blood and bone marrow involvement, which is usually observed in older people. Genetic mutations, epigenetic changes and miRs play a role in CLL pathogenesis. Del 11q, dell17q, del6q,trisomy 12, p53 and IgVH mutations are the most important genetic changes in CLL. Deletion of miR-15a and miR-16a can increase bcl2 gene expression, miR-29 and miR-181 deletions decrease the expression of TCL1, and miR-146a deletion prevents tumor metastasis. Epigenetic changes such as hypo- and hypermethylation, ubiquitination, hypo- and hyperacetylation ofgene promoters involved in CLL pathogenesis can also play a role in CLL. Expression of CD38 and ZAP70, presence or absence of mutation in IgVH and P53mutation are among the factors involved in CLL prognosis. Use of monoclonal antibodies against surface markers of B-cells like anti-CD20as well as tyrosine kinase inhibitors are the most important therapeutic approaches for CLL.

In-Vitro Drug Sensitivity Screening in Chronic Lymphocytic Leukemia (CLL) Primary Patient Samples Identifies Drug Candidates for Precision Cancer Therapy

Abstract Introduction Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults and is currently considered incurable. Although current treatment regimens prolong life for patients, CLL eventually relapses. Efficient therapies may require a personalized approach that combines targeting cancer cells and the tumor microenvironment by restoring the patient's own anti-tumor immunity. However, a major limitation is that no efficient approach exists to identify the most effective drugs for each patient and cancer stage. With the aim to support future introduction of individualized treatment for patients, we assessed the sensitivity of CLL patient samples to several drug candidates using our in vitro functional drug sensitivity screening platform. Methods We have established novel in vitro culture settings that mimic the CLL tumor microenvironment and allow proliferation of CLL cells for 5 days. Using our unique method, we performed drug screening on 26 patient samples and 10 healthy donors against a customized, annotated library of 516 drugs including kinase inhibitors, proteasome inhibitors, B-cell pathway inhibitors and several other approved drug classes. Primary patient samples were cultured in 384 well-plates with the presence of individual drugs over a concentration range over 5 logs. Drug sensitivity was assessed using CellTiter-Glo® luminescent cell viability assay and CellTox™ green cytotoxicity assay on day 5. Drug Sensitivity Score (DSS) was then calculated for each drug using the IC50 value, slope and area under the curve (AUC). DSSs for CLL patient samples were next compared with DSS of healthy donors for the full patient sample cohort screened so far to generate a selective DSS (sDSS = DSSpatient - DSShealthy) for each patient. Drugs which have sDSS &gt;5 were considered clearly more effective for patient samples in the in vitro test system. CLL samples were assessed for sDSS using our screening data and we ranked all the drugs by their score. Results In order to find drug candidates for targeted therapies in CLL patients, we performed in vitro drug sensitivity screening on 13 IgVH unmutated and 13 IgVH mutated CLL patient samples, as well as 10 healthy donors (due to the lower number of cells healthy donors were pooled into two samples of 5 donors each). Our in vitro assay showed that proteasome inhibitors, kinase inhibitors and several approved CLL drug candidates were considered sensitive in the majority of patient samples. This included venetoclax, the Bcl-2 inhibitor ABT-737, doxorubicin, acalabrutinib, other kinase inhibitors (sunitinib, volasertib, trametinib, copanlisib) and proteasome inhibitors (carfilzomib, bortezomib). Selective drug sensitivity scores of the top 5 drugs in all patient samples (73 drugs in total) are shown in the heatmap (Figure 1). Venetoclax showed a higher sDSS score in 10 of the 20 patients with an average sDSS score of 22.3 followed by ABT-737 (Bcl-2 inhibitor) with an average sDSS score of 19.7. By performing hierarchical clustering analysis (Euclidean distance, Ward linkage method), we observed unsupervised clustering of patient samples irrespective of the IgVH mutation status. We are currently expanding the analysis by classifying the patient samples by age, sex and mutation status. Conclusion Our novel CLL culture method that allows cell proliferation along with our established functional in-vitro drug sensitivity screening platform enabled us to screen a number of patient samples and evaluate the sensitivity of a library of approved drugs and investigational drug candidates for CLL. Our analysis shows that several drugs may be effective for CLL and can be tested in drug combinations in order to identify synergistic effects. As a future perspective, we want to combine machine learning strategies with the experimental drug screening strategies to identify drug combinations and validate drug candidates by xenografting and in precision medicine clinical trials. Figure 1: Selective Drug Sensitivity Screening (sDSS) score for top 3 drugs (44 drugs) for 20 CLL patient samples. Green label is IgVH mutated CLL patient samples and blue label is IgVH unmutated patient samples. Disclosures Schjesvold: Oncopeptides: Consultancy; Abbvie: Honoraria; Novartis: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Adaptive: Consultancy; Bayer: Consultancy; Bristol Myers Squibb: Consultancy; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding.

Integrated Proteomic and Phosphoproteomic Analysis Reveal Novel Targets and Suggest Rationale for Ibrutinib Efficacy in UM-CLL

Integrated Proteomic and Phosphoproteomic Analysis Reveal Novel Targets and Suggest Rationale for Ibrutinib Efficacy in UM-CLL

Real Life Dates of Chronic Lymphocytic Leukemia (CLL) in Germany. Interim Analysis of 234 Consecutively New Diagnosed Patients in a German Epidemiological-Clinical Register

Background:Therapy studies yield important insights into clinical features and therapeutic options of CLL. However studies in Germany represent less then 20% of all CLL patients (pts); pts >75 years (yrs) are included in 3% only. Furthermore initial phases are not reflected in multicenter studies and data representing real life situation are scarce.Methods:Out of 1.443 patients consulting our center because of leucocytosis (>10 Gpt/l) between 2003/07/01 and 2018/06/30 we diagnosed CLL consecutively in 234 pts (16,2%): 143 male (m), 91 female (f). Median age at diagnosis is 71,4 yrs. Median leucocyte count is 18,0 Gpt/l. BINET - Stages are: A in 82%, B in 11% and C in 7% of pts. Diagnoses are strictly based on WHO definition: Pts with small lymphocytic lymphomas (sLL, n=11) are included but those with monoclonal lymphocytosis of unknown significance (MLUS) or monoclonal B - lymphocytoses with uncertainly flow cytometry (FC) results are excluded. Histopathology, FC and genetics have been performed by external hematological reference laboratories. All investigations followed the rules of best clinical and laboratory practice. Dates of death are given by the record sections of the involved communities (deadline 2018/06/30).To address different questions we defined 3 groups of pts: A: “collective group”, (n=234), i.e. all pts.diagnosed from 2003/07/01 to 2018/06/30. B: “epidemiological group” (n=129), i.e. pts from 3 communities (113.000 inhabitants - inc - in 2009/12/31) in close proximity referred to our center. C: “genetic features group” (n=99), i.e. pts diagnosed continuously between 2012/07/01 and 2018/06/30 with systematically performed genetics, (i.e. at least 80% of pts in this group. Genetics are: Cytogenetic (banding) n=88; FISH (del 6q21/6q, del 11q22.3, +12/+12q, del 13q14/ 13q34, del 17p13.1, 14q32) n=85. PCR (IgVH status - mutated vs. unmutated; TP53, NOTCH1, SF3B1 mutation) n=82.Results:“Collective group” (A): 7,5 years OS is 72% and 15 years OS is 33% respectively (KAPLAN-MEIER). Age of pts acts as a predominant factor for long term OS: 77% in pts < 60 yrs vs. 40% in pts ≥60 to <75 yrs vs. 16% in pts ≥75 yrs. 15 yrs OS of treated pts is 32% (n=78, median age at diagnosis: 73,3 yrs, m : f = 1,50); 15 yrs OS of untreated patients is 33% (n=146, median age at diagnosis: 71,0 yrs, m : f = 1,56). Mean time to 1st treatment was 16,0 month (0,1 to 132,6 months).“Epidemiological group” (B): Raw incidence is 7,56/105 inh. (m: 9,13, f: 6,06), age adjusted incidence is 10,3/105 inh (m: 12,5, f: 8,3), with highest rate of age between 75 to < 80 yrs. Standard incidence is calculated as 6,27/105 (BRD 1987), 4,78/105 (Europe) and 5,06/105 (USA 2000). Prevalence could be measured directly (2017-12-31): 79,1/105 inh.“Genetic features group” (C): IgVH mutation status is hypermutated in 48 pts. vs. unmutated in 39 pts. Results of FISH analyses (n = 85) are: del 6: 5,5%, del 11: 17,6%, +12: 3%, del 13q: 70,3%, del 17p: 8,8%; normal status: 13,2%. PCR revealed mutations of NOTCH1 in 7%, of SF3B1 in 10,5% and of TP53 in 8,6% of 82 pts, respectively. Combined del17p by FISH and molecular TP53 mutations at diagnosis are found in 3 of 81 pts (3,7%).Cytogenetic findings different to results of FISH and/or PCR were found in 36 of 82 pts (43,9%).Calculated 5 yrs freedom of therapy is 75% in IgVH hypermutated pts vs. 45% in unmutated pts (p = 0,0005; log rank test); calculated 5 yrs OS is 92% vs. 59% (p=0,023).Special situations in CLL: 13 of 234 pts (5,5%) have one parent or sibling with low grade NHL, mostly CLL. In contrast there is non spouse suffering of CLL / low grade NHL. Deficiency in Immunoglobuline G (Ig G < 4,0 g/l) was found in 17 of 199 pts at diagnose (8,5%), 29 pts (12,4%) were substituted with Ig G's in the Course of CLL. In 14 (6,0%) pts. we diagnosed a 2nd hematologic malignancy (3x cMPN, 1x CML, 3x MDS, 3x AML, 1x FL, 1x MCL, 1x MZL, 1x DLCBL-independent of CLL; monoclonal gammopathies are not considered). RICHTER syndrome was found in 1 case, but progression to plasmocytic/plasmoblastic disease was seen in 3 cases. Li FRAUMENI Syndrome was revealed in 1 male patient, he has been in stable disease for 13 yrs.Conclusion:CLL seems to be more frequent than yet considered. IgVH mutation status seems to be very important as a single prognostic factor concerning time of 1st therapy as well as OS. Clinical features of CLL are very different and impressing. DisclosuresBöttcher:Genentech: Research Funding; Janssen: Honoraria; AbbVie: Honoraria, Research Funding; Celgene: Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Subtype assignment of CLL based on B-cell subset associated gene signatures from normal bone marrow - A proof of concept study.

Diagnostic and prognostic evaluation of chronic lymphocytic leukemia (CLL) involves blood cell counts, immunophenotyping, IgVH mutation status, and cytogenetic analyses. We generated B-cell associated gene-signatures (BAGS) based on six naturally occurring B-cell subsets within normal bone marrow. Our hypothesis is that by segregating CLL according to BAGS, we can identify subtypes with prognostic implications in support of pathogenetic value of BAGS. Microarray-based gene-expression samples from eight independent CLL cohorts (1,024 untreated patients) were BAGS-stratified into pre-BI, pre-BII, immature, naïve, memory, or plasma cell subtypes; the majority falling within the memory (24.5–45.8%) or naïve (14.5–32.3%) categories. For a subset of CLL patients (n = 296), time to treatment (TTT) was shorter amongst early differentiation subtypes (pre-BI/pre-BII/immature) compared to late subtypes (memory/plasma cell, HR: 0.53 [0.35–0.78]). Particularly, pre-BII subtype patients had the shortest TTT among all subtypes. Correlates derived for BAGS subtype and IgVH mutation (n = 405) revealed an elevated mutation frequency in late vs. early subtypes (71% vs. 45%, P < .001). Predictions for BAGS subtype resistance towards rituximab and cyclophosphamide varied for rituximab, whereas all subtypes were sensitive to cyclophosphamide. This study supports our hypothesis that BAGS-subtyping may be of tangible prognostic and pathogenetic value for CLL patients.

Additional value of quantitative expression of ZAP-70 and CD38 for prognostic factors in chronic lymphocytic leukemia.

e19006 Background: Chronic lymphocytic leukemia (CLL) has heterogeneous clinical course. Identification of prognostic markers is important for proper management of disease. Cytogenetics, IgVH mutation status, TP53 functional status, CD38, ZAP-70 and CD49d expression are accepted prognostic factors in CLL. ζ-chain-associated protein kinase 70 (ZAP-70) is associated with disease progression. However, measurement of ZAP-70 and CD38 expression relies on qualitative flow cytometry. Authors tested hypothesis whether quantitative analysis of markers can improve information about prognosis of CLL. Methods: 217 CLL patients were included; diagnosis was made from peripheral blood by morphology and immunophenotyping by flow cytometry with common antibodies. Expressions of intracellular ZAP-70 and surface CD38 were measured using quantitative fluorescence cytometry. Standardized fluorescent microparticles were used to quantify molecules of equivalent soluble fluorochrome (MESF units). Analysis of rearrangement of the IgVH gene was performed according to the European recommendation. Deletions at 11q22-q23 ( ATM), 17p13 ( TP53), 13q34 ( RB1) loci and trisomy of chromosome 12 were detected by I-FISH. Results: Patients’ ages ranged from 33 to 86 years and included all Rai stages. Expression of ZAP-70 ranged from 546 to 5,955 MESF, CD38 from 1,886 to 31,619 MESF. 48% patients embodied mutated and 52% unmutated IgVH status. In cytogenetics, del(13q) was detected in 59%, trisomy 12 in 13%, del(11q) in 21%, and del(p53) in 8% of patients. IgVH (in %) was plotted against ZAP-70 (in MESF) and patients were divided into high (HR) and low risk (LR) groups by cluster analysis based on Kaplan-Meier overall survival curves. Patients in HR group had significantly poorer prognoses (p = 0.0001) than those in LR group. When patients from HR group were clustered according to CD38 expression, very high risk (VHR) group with high CD38 and significantly worse prognosis (p = 0.035) was identified. Conclusions: We have demonstrated that quantitative ZAP-70 and CD38 expressions cannot replace IgVH analysis, but, contrary to that, addition of quantitative data can further specify prognoses of patients with CLL.

Immunogenetics of chronic lymphocytic leukemia.

Cytogenetic aberrations as well as presence of IGVH mutations are the underlying reason for clinical heterogeneity in Chronic Lymphocytic Leukemia (CLL). The presence of IGVH mutations as well as the predominant gene usage shows geographical variations. However, there is no study from India addressing immunogenetics of CLL. In a first Indian study we document the immunogenetics of CLL in a large tertiary hospital. We analyzed IGVH mutation status, VH gene usage, cytogenetic abnormalities using FISH, immunophenotyping data and correlated them with standard clinical variables in 84 patients of CLL. Advanced Rai stage (Stage 3/4) was seen in 45% of our patients, where as 13q deletion was the commonest clonal cytogenetic abnormality detected in 48.4% of the cases. IGVH unmutated cases (55.2%) showed higher proportion expressing CD38 and CD49d, a preferential usage for VH1 and VH3 families (55.2%), presentation at an advanced Rai stage (52.8%) as well as more frequent presence of p53 deletions. As compared to the IGVH mutated cases greater proportion of IGVH unmutated patients (70%) required treatment. However, there was no significant difference in the time to treatment between mutated and unmutated cases which can be attributed to relatively short median follow up of 10 months. To summarize, we have seen a higher proportion of IGVH unmutated patients in our cohort (55.2%). The commonly used VH genes in the Indian population are IGVH 2-5, IGVH 1-2 and IGVH 1-69. Longer clinical follow up and a larger cohort is necessary to confirm the prognostic value of IGVH mutation analysis in Indian Patients with CLL.

Genomic data integration in chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (CLL) is a heterogeneous disease and the most common adult leukemia in western countries. IgVH mutational status distinguishes two major types of CLL, each associated with a different prognosis and survival. Sequencing identified NOTCH1 and SF3B1 as the two main recurrent mutations. We described a novel method to clarify how these mutations affect gene expression by finding small-scale signatures that predict the IgVH, NOTCH1 and SF3B1 mutations. We subsequently defined the biological pathways and correlation networks involved in disease development, with the potential goal of identifying new drugable targets. We modeled a microarray dataset consisting of 48807 probes derived from 163 samples. The use of Fisher's ratio and fold change combined with feature elimination allowed us to identify the minimum number of genes with the highest predictive mutation power and, subsequently, we applied network and pathway analyses of these genes to identify their biological roles. The mutational status of the patients was accurately predicted (94-99%) using small-scale gene signatures: 13 genes for IgVH, 60 for NOTCH1 and 22 for SF3B1. LPL plays an important role in the case of the IgVH mutation, whereas MSI2, LTK, TFEC and CNTAP2 are involved in the NOTCH1 mutation, and RPL32 and PLAGL1 are involved in the SF3B1 mutation. Four high discriminatory genes (IGHG1, MYBL1, NRIP1 and RGS1) are common to these three mutations. The IL-4-mediated signaling events pathway appears to be involved as a common mechanism and suggests an important role of the immune response mechanisms and antigen presentation. This retrospective analysis served to provide a deeper understanding of the effects of the different mutations in CLL disease progression, with the expectation that these findings will be clinically applied in the near future to the development of new drugs.

FAT1 Mutations Influence Time to First Treatment in Untreated CLL

Introduction:Despite mapping of the mutational landscape in patients (pts) with chronic lymphocytic leukemia (CLL), there remains limited information regarding the functional or clinical impact of less common, recurrently mutated genes. Over the past several years, the influence of high-risk mutations has been reported. With the advent of readily available next generation sequencing (NGS), physicians receive a large amount of genomic data, many times without information regarding the clinical impact of less validated, mutations. Using results from a commercially available NGS platform, we identified a relatively high prevalence of mutations in the gene, FAT1, in treatment naïve (TN) CLL pts.FAT1 encodes a protein in the cadherin superfamily, important in regulating WNT signaling, with a role in tumor suppression. Aberrant mutated FAT1 expression has been associated with acute lymphoblastic leukemias with an intermediate clinical course. Loss of protein, through chromosomal deletions has been implicated in many solid tumors and associated with tumor progression. The majority of mutations arise in the cadherin repeats, previously shown to diminish antagonism of β-catenin, allowing its nuclear localization (Morris Nat Genetics 2013). In CLL, FAT1 mutations have been demonstrated to enrich in fludarabine refractory pts (10.3%) but with a relatively low prevalence in untreated pts (1.1%) (Messina, Blood 2014). Given a high prevalence of FAT1 mutations in our database and evidence suggesting mutated FAT1 contributes to tumor evolution, we investigated the clinical impact of clonal FAT1 mutations in pts with CLL.Methods:Patients were identified for inclusion if mutational analysis was performed prior to, or within 12 months of treatment start date. Only pts treated initially with a signal transduction inhibitor (STI) were included. Treated pts were either on protocol or received their STI as standard of care. All pts underwent whole exome profiling with a lymphoid specific NGS panel, including 75 genes (Genoptix Inc.). Analysis was performed on peripheral blood or bone marrow aspirates. The primary outcome was time to first treatment (TTFT). The log-rank test was used to compare Kaplan-Meir curves. Double-sided P values <0.05 were considered significant. Fischer exact test was used to compare baseline characteristics. All calculations were performed using Prism V6.0.Results:In total we analyzed 172 pts, 118 (69%) remain on observation, 54 (31%), have been treated at data cutoff. Nineteen (11%) pts were found to be FAT1 mutated (mFAT1) and 153 (89%) were FAT1 wild type (wtFAT1). The median time from diagnosis to mutational profiling for the cohort was 35 months (range 0-301). We identified 21 total mutations, 17 unique (82% cadherin domain, 18% EGF like region). Recurrent mutations were identified, each occurring in 2 pts (p.A636T, p.P1614L, p.R1257Q, p.R2041H). One pt had an indel and 1 pt had an inframe deletion, the remaining were missense. We found no significant differences between groups in regards to age, IGVH mutation status or co-occurrence of high-risk mutations in NOTCH1, SF3B1, TP53 or ATM. Only 17p deletions occurred significantly more in mFAT1 pts (24%) vs. wtFAT1 (7%), p=0.04, other cytogenetic abnormalities were balanced. mFAT1 pts had significantly shorter TTFT, 50 versus 143 months respectively, p=0.02. We investigated if mFAT1 status could stratify IGVH mutated pts but found no differences in TTFT. Eleven mFAT1 pts have received treatment (82% ibrutinib, 18% acalabrutinib), 8 remain on observation. All mFAT1 pts have responded.Conclusions:Given the increasing use of rapidly available NGS testing, physicians receive genomic information with limited data regarding clinical impact. We identified a higher prevalence of FAT1 mutations in TN pts, than that previously reported. FAT1 mutations associated with deletion 17p but not other high-risk genetic mutations. Recurrent FAT1 mutations were identified and commonly found in the cadherin domain. mFAT1 pts have a significantly shorter TTFT than wtFAT1 pts. When treated with novel agents, there was no difference in response rates. These findings suggest FAT1 mutations in TN pts may be more common than previously reported and identify an intermediate risk for progression. Additional studies investigating the influence of recurrent FAT1 mutations and association with 17p deletions in TN pts are warranted. DisclosuresAllan:Pharmacyclics: Speakers Bureau.

Dynamic Alterations in Gene Expression in Ibrutinib Treated CLL Reveal Profound Impact on Multiple Signaling Pathways

Ibrutinib covalently binds Bruton tyrosine kinase (BTK) leading to sustained target inhibition. On-target effects include reductions in BCR and NF-κB signaling and inhibition of tumor proliferation and survival (Herman, Blood 2014). In randomized trials, ibrutinib induced superior responses and extended the survival of patients with chronic lymphocytic leukemia (CLL) in both the relapsed/refractory and frontline setting (Byrd, NEJM 2014; Burger, NEJM 2015). However, deep remissions are uncommon and most patients have readily detectable residual disease in the blood. To better understand the biology of persistent disease on ibrutinib, we performed RNA-sequencing on CD19+ selected tumor cells from 14 of 86 patients treated with single-agent ibrutinib (ClinicalTrials.gov NCT01500733); 8/14 (57%) had an unmutated IGVHsequence, 8/14 (57%) had del(17p), 6/14 (43%) were previously untreated and 4/14 (29%) had relapsed on ibrutinib at the time of analysis (range: 8-47 months). At best response, 3/14 (21%) achieved a CR, 10/14 (71%) a PR or PRL and 1/14 (7%) had SD. Three samples per patient were analyzed: T1 (baseline), T2 (day 28) and T3 (6 months), for a total of 42 samples. Principal component analysis (PCA) grouped samples by patient, as observed in a similar study (Herishanu, Blood 2011). After adjusting for patient-specific effects, PCA clearly revealed the effect of treatment, separating on-treatment samples (T2, T3) from baseline (T1). To identify differentially expressed (DE) genes, we selected transcripts that showed at least a 2-fold change between baseline and treatment samples (FDR<0.1). Of the ~14,000 measured genes, there were 653 DE genes on-treatment compared to baseline. Notably, T3 samples demonstrated stronger changes in gene expression than T2 samples, in line with a more profound treatment response with continued drug exposure. Compared to T1, there were 80 DE genes expressed at T2 only, 330 at T3 only, and 243 at both T2 and T3. Among all DE genes, 498 decreased and 155 increased at either T2 or T3 compared to T1.To identify the biologic impact of treatment, we dissected the gene expression changes using pathway-specific gene signatures represented in the KEGG pathways with the molecular signatures database tool (MSigDB; Broad Institute, Boston, MA). Strikingly, no pathways were significantly enriched on treatment. In contrast, multiple gene signatures (n=46) were significantly turned down on ibrutinib (FDR<0.01). Using more stringent cutoffs (p<0.0005; FDR q<0.005; ≥5 significant genes per pathway), nine KEGG pathways were significantly inhibited by ibrutinib, including cytokine signaling, cell adhesion, systemic lupus, p53 response, MAPK signaling, cell cycle, focal adhesion, calcium signaling, and Wnt signaling. We also applied the same analysis to experimentally-derived B cell-specific gene signatures (Shaffer, Immunity 2001; Herishanu, Blood 2011), demonstrating significant inhibition of both BCR and NF-κB signaling pathways. We derived signature scores by averaging the expression of the signature-specific genes and estimated the degree of pathway inhibition from the ratio of signature averages in treated versus baseline samples. Of the 11 significantly altered pathways, all demonstrated >50% reduction in signature scores, ranging from a 71% reduction for BCR to 54% for p53. The degree of inhibition of the signaling pathways was independent of biologic and clinical features, including IGVH mutation status, presence or absence of del(17p), and prior treatment status.In summary, ibrutinib induces widespread changes in gene expression in CLL cells in vivo reflecting strong inhibitory effects across multiple pathways that suggest a broader impact on tumor biology than expected from pure loss of BTK function. The surprising extent of changes across multiple diverse pathways could reflect inhibition of tumor-microenvironment crosstalk and consequent changes in the tumor milieu. This notion is consistent with the more profound inhibition observed with longer treatment duration. Equally notable, we found no distinct biologic signature(s) upregulated in response to ibrutinib which may indicate the absence of a cellular stress response and is consistent with the apparent resting state of the residual tumor and the observed slow attrition of cells over time.This research was supported by the Intramural Research Program of the NHLBI. DisclosuresFarooqui:Merck: Employment. Wu:Neon Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder. Wiestner:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding.

The Role of HOXA4 in Chronic Lymphocytic Leukaemia Progression and Response to Therapy

Chronic lymphocytic leukaemia (CLL) is the most common form of adult leukaemia worldwide. Patients display a highly variable clinical course, with some requiring immediate therapeutic intervention while others can remain untreated for years. We have previously reported that DNA methylation of the homeobox A4 (HOXA4) promoter can serve as part of a three gene prognostic signature with CD38 and BTG4 to predict time to first treatment (TTT) in Stage A patients. HOXA4 encodes a transcription factor that is expressed in haematopoietic progenitor cells and is involved in embryonic development and B-cell differentiation, and its aberrant epigenetic regulation has been identified in multiple forms of leukaemia. In this study we have sought to elucidate the role of HOXA4in the progression of CLL and determine the functional consequences of its expression.We analysed DNA methylation of the HOXA4 promoter by pyrosequencing in a heterogeneous cohort of 163 CLL patients (median age: 70; median follow-up: 10 years), of whom 60% were Binet Stage A, 16% Stage B and 24% Stage C. Data was collected regarding treatment history, TTT and overall survival, as well as cytogenetic abnormalities and IGVH mutation status. HOXA4 methylation increased with disease progression and was significantly higher in Stage C patients (median 74%) than those with Stage A (62%; p = 0.03) and Stage B disease (65%; p < 0.05). HOXA4 methylation was positively correlated with IGVH sequence homology (r = 0.34, p < 0.0001) and negatively associated with TTT among patients who have started chemotherapy (p = 0.04) and with overall survival (p = 0.04). No associations were observed between HOXA4 methylation and 11q, 13q or 17p deletions, or TP53 and ATMmutations.To investigate the role of HOXA4 in the evolution of the disease, we analysed samples taken at multiple timepoints from 42 patients, of whom 29 were undergoing treatment and 13 remained untreated. HOXA4methylation significantly increased in patients undergoing treatment (p = 0.01), but did not differ in untreated patients (p = 0.19).We hypothesised that silencing of HOXA4 may be selected for during treatment due to its expression conferring increased sensitivity to chemotherapy. Using a lentiviral system, we observed that re-expression of HOXA4increased drug sensitivity in a malignant differentiated B-cell line (Raji). Significantly higher apoptosis was identified after treatment with 3 μM and 10 μM fludarabine (both p < 0.001) and 1 μM and 10 μM ibrutinib (p < 0.01 and p < 0.001), but not 1 μM and 10 μM idelalisib.To confirm the translational relevance our observations, we overexpressed HOXA4 in primary CLL cells derived from four patients and confirmed increased apoptosis in response to 3 μM and 10 μM fludarabine treatment in comparison to control cells (p = 0.02 and p < 0.01). Further work is underway in primary CLL cells to elucidate the pathways under the control of HOXA4 that may confer this drug sensitivity.Our ongoing work may indicate that HOXA4 is also implicated in the progression of CLL through directing malignant cells to the protective bone marrow niche, thereby further reducing sensitivity to antimetabolites. In cell lines HOXA4 up-regulates the expression of RGS2 and RGS16, which are negative regulators of the CXCR4-CXCL12 signalling axis, and we have identified selection for biallelic HOXA4methylation in primary acute lymphoblastic leukaemia cells following engraftment in mice (median in primary cells: 80%; engrafted cells: 92%; p < 0.0001).To determine the origins of HOXA4 dysregulation during the course of the disease, we analysed prospective blood samples from the European Prospective Investigation into Cancer and Nutrition (EPIC) from 20 individuals diagnosed with CLL up to 17 years after blood draw (median: 7 years) and 20 age-matched controls who remained free of cancer. We observed that HOXA4methylation was significantly higher among future CLL patients (median: 49% vs 42%; p = 0.01) and was inversely correlated with time to diagnosis, but did not reach statistical significance (r = -0.39, p = 0.09).Together, our findings suggest that silencing of the HOXA4 gene is an early event in CLL which is selected for during the course of disease through reduced sensitivity to chemotherapeutic agents. Our ongoing work will identify downstream targets that may be implicated in conferring sensitivity, and which may serve as biomarkers to predict prognosis and inform treatment strategies. DisclosuresJunge:AstraZeneca: Other: Salary, Research Funding.