Notch2 Increases the Resistance to Venetoclax-Induced Apoptosis in Chronic Lymphocytic Leukemia B Cells by Inducing Mcl-1

Chronic lymphocytic leukemia (CLL) has experienced a clinical revolution—thanks to the discovery of crucial pathogenic mechanisms. CLL is still an incurable disease due to intrinsic or acquired resistance of the leukemic clone. Venetoclax is a Bcl-2 inhibitor with a marked activity in CLL, but emerging patterns of resistance are being described. We hypothesize that intrinsic features of CLL cells may contribute to drive mechanisms of resistance to venetoclax. We analyzed the expression of Interferon Regulatory Factor 4 (IRF4), Notch2, and Mcl-1 in a cohort of CLL patients. We evaluated CLL cell viability after genetic and pharmaceutical modulation of Notch2 expression in patients harboring trisomy 12. We tested venetoclax in trisomy 12 CLL cells either silenced or not for Notch2 expression or in combination with an inhibitor of Mcl-1, AMG-176. Trisomy 12 CLL cells were characterized by low expression of IRF4 associated with high levels of Notch2 and Mcl-1. Notch2 and Mcl-1 expression determined protection of CLL cells from spontaneous and drug-induced apoptosis. Considering the involvement of Mcl-1 in venetoclax resistance, our data demonstrated a contribution of high levels of Notch2 and Mcl-1 in a reduced response to venetoclax in CLL cells carrying trisomy 12. Furthermore, reduction of Mcl-1 expression by silencing Notch2 or by treatment with AMG-176 was able to restore the response of CLL cells to venetoclax. The expression of Notch2 identifies a subset of CLL patients, mainly harboring trisomy 12, characterized by high levels of Mcl-1. This biological mechanism may compromise an effective response to venetoclax.

Detection Rate of Integrated Molecular and Cytogenetic Biomarker Testing for Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in western countries and typically occurs in elderly individuals. There will be an estimated 21,250 newly diagnosed cases in the United States this year and 4,320 deaths. Due to the highly variable clinical course of this disease, prognostication and risk stratification methods are necessary for guiding decisions on clinical management. Integrated prognostic models incorporating laboratory testing for multiple molecular, cytogenetic, and other biomarkers have recently been proposed by major clinical guidelines to classify patients into risk subgroups. The current NCCN Guidelines for Chronic Lymphocytic Leukemia/Small Lymphocytic Leukemia describe such an integrated prognostic model known as the Rossi model that includes TP53, NOTCH1, SF3B1, and BIRC3 mutations along with the cytogenetic abnormalities detected by fluorescence in situ hybridization (FISH) to classify CLL patients into 4 distinct prognostic subgroups: high-risk (TP53 and/or BIRC3 abnormalities), intermediate-risk (NOTCH1 and/or SF3B1 mutations and/or deletion 11q), low-risk (trisomy 12 and wild-type for all genetic lesions), and very low-risk (deletion 13q only). The 10-year survival rates for these subgroups are 29%, 37%, 57%, and 69%, respectively. To assess the clinical value of an integrated biomarker testing approach, we analyzed results of 651 consecutive cases submitted to our clinical diagnostic laboratory for testing on our integrated panel of molecular and cytogenetic biomarkers for CLL. Our panel includes detection of genomic alterations by FISH (deletion 6q, 13q, 11q, 17p, trisomy 12, IGH rearrangement, and IGH/CCND1 translocation) and detection of sequence variants in BIRC3, BTK, MYD88, NOTCH1, PCLG2, SF3B1, and TP53 by next-generation sequencing (NGS). In total, 472 cases had positive findings by either FISH (90%) or NGS (46%) for a detection rate of 72.5%. Using the Rossi integrated prognostic model, 17.5% of cases fell into the high-risk subgroup, 20% of cases fell into the intermediate-risk subgroup, 43.5% of cases fell into the low-risk subgroup, and 19% of cases fell into the very low-risk subgroup. Importantly, among cases with positive FISH findings, 40.1% of cases also had positive molecular findings. In approximately 84% of cases belonging to the low-risk cytogenetic subgroups by FISH assessment alone, the incorporation of molecular findings resulted in reclassification into a higher-risk subgroup. Among the FISH-negative cases, 17% were classified as high-risk or intermediate-risk based on the molecular findings. Together, these findings support the clinical value of an integrative biomarker testing approach that includes both molecular and cytogenetic biomarkers to stratify CLL patients into risk subgroups to help guide decisions on clinical management. Disclosures Prathapam: Quest Diagnostics: Current Employment. Maharjan: Quest Diagnostics: Current Employment. Powers: Quest Diagnostics: Current Employment. Freitas: Quest Diagnostics: Current Employment. Sun: Quest Diagnostics: Current Employment. Tan: Quest Diagnostics: Current Employment. Gupta: Quest Diagnostics: Current Employment. Hucthagowder: Quest Diagnostics: Current Employment. Graham: Quest Diagnostics: Current Employment. Whitman: Quest Diagnostics: Current Employment. Khadgi: Quest Diagnostics: Current Employment. Daniel: Quest Diagnostics: Current Employment. Racke: Quest Diagnostics: Current Employment. Champion: Quest Diagnostics: Current Employment.

A Study of Ribonucleotide Reductase mRNA Expression and Its Prognostic Role in Patients with Chronic Lymphocytic Leukemia

Background Ribonucleotide Reductase (RNR) is responsible for converting ribonucleotides to deoxyribonucleotides required for DNA replication and repair. RNR consists of two subunits, termed subunit 1 (RRM1) and 2 (RRM2). Imbalance in the regulation of RNR activity and control of dNTPs' pool leads to genomic instability and increases mutation rate. RNR expression has been associated with prognosis in pancreatic, non-small-cell lung, breast, and biliary tract cancer. However, RNR expression in chronic lymphocytic leukemia (CLL) and its possible prognostic role have not been investigated yet. Aim In this study we evaluate the possible prognostic role of RNR expression in CLL. Method The study comprised patients with immunophenotypically confirmed disease at the time of sample collection. Peripheral whole blood samples were collected from 84, 27, 15, and 9 patients before treatment, after one, two, and three lines of treatment respectively. RNA extraction and reverse transcription were carried out using standard protocols. A Taqman based real-time PCR was performed on a CFX96 RT-PCR system (Bio-Rad Laboratories, Hercules, CA, USA). For both the housekeeping and target genes, a Taqman primer/probe mix was used according to the manufacturer's instructions (Applied Biosystems, Foster City, CA, USA). RRM1 and RRM2 mRNA levels were expressed as an RRM1-2/GAPDH ratio. Western blot analysis was performed to quantify the RRM1 protein levels in a random sample of 41 patients. Antibodies used were: RRM1 #3388, β-actin #4967 and anti-rabbit IgG HRP-conjugated #7074 (Cell Signaling Technology, Danvers, MA, USA). Detection was done using the ECL western blotting reagents. Statistical analysis was conducted to study the possible correlations between the variables. All reported p values are two-tailed. Statistical significance was set at p<0.05 and analyses were conducted using SPSS statistical software (version 22.0). Results From 135 CLL patients included in the study 56.3% were female and the median age at diagnosis was 64 years. Peripheral blood was collected in 84 treatment-naïve patients (62.2%). Median follow up was 6.66 years (3.47 ─ 11.13) and median time from diagnosis until 1st line treatment was 23.1 months (IQR: 5.8 - 56.5 months). Out of 135 patients, 69 (51,1%) received 1 st line treatment and 35 patients (25,9%) 2 nd line treatment with median time between the two treatment lines being 26.5 months (IQR: 7.8 - 40.8 months). Furthermore, 48.5%, 33.8%, 12.3%, 3.1% and 2.3% of the patients had Rai score 0, I, II, III, IV respectively. The median mRNA expression of RRM1 was 0.04 (IQR: 0 - 0.09) and of RRM2 was 0.01 (IQR: 0 - 0.1). RRM1 mRNA expression was significantly higher in patients without anemia (p=.025) and without lymphadenopathy (p=.002). Higher values of ESR (r=-.30; p=.028), LDH (r=-.20; p=.026) and Rai score (r=-.18; p=.037) were associated with lower expression of RRM1 mRNA. In addition, TP53 gene deletion detected by FISH was associated with higher RRM1 mRNA expression (p=.036). Significantly higher RRM2 mRNA expression was reported in patients without lymphadenopathy (p=.021) and Rai score 0 (p=.003). Moreover, higher was the expression of RRM2 mRNA in cases with Trisomy 12 (p=.050). In samples collected before treatment, higher values of RRM1 mRNA expression were statistically significantly associated with lower RAI score (r=-.30; p=.005) and longer time periods between the first two lines of treatment (r=.95; p=.050). Western blot analysis confirmed detection of RRM1 protein but statistical correlation was not carried out due to lack of material from the whole group of patients. Conclusion For the first time, mRNA expression of RRM1 and RRM2 is studied in patients with CLL. These results show RNR involvement in the pathophysiology of CLL. RRM1 and RRM2 mRNA higher expression found in 17p deletion and trisomy 12 cases respectively may be consistent with the existence of a methylation-depended mechanism proposed by other studies. Therefore, these results demonstrate RNR's potential role as a prognostic factor, and make it a probable therapeutic target. A study including a larger number of cases could further confirm our results. Figure 1 Figure 1. Disclosures Kyrtsonis: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/Genesis Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria; Sanofi: Membership on an entity's Board of Directors or advisory committees. Panagiotidis: Abbvie: Research Funding; Pfizer: Research Funding; Janssen: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Takeda: Research Funding; Sandoz: Research Funding; Bristol-Myers Squibb: Research Funding; Roche: Research Funding; Astellas: Research Funding. Viniou: Sandoz: Research Funding; Takeda: Research Funding; Novartis: Honoraria, Research Funding; Sanofi: Research Funding; Janssen: Honoraria, Research Funding; Pfizer: Research Funding; Abbvie: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Roche: Research Funding; Astellas: Research Funding; Celgene: Research Funding.

NOTCH1 Mutation in CLL Enhances Cell Cycle G1/S Transition through Specific Cyclin Overexpression: Preclinical Ground for CDK4/6 Targeted Inhibition

Introduction: The partially understood biological consequences of the NOTCH1 acquired lesion, seems to be distinctive enough among chronic lymphocytic leukemia (CLL) patients, as clinical studies have repeatedly found specific features: intermediate prognosis, anti-CD20 poorer responses, and a higher frequency of trisomy 12 and Richter transformation. Though located in a different domain, the activating nature of NOTCH1 mutation in T lymphoblastic leukemia relies on cell cycle regulators. In fact, pivotal studies, from the pre-next generation sequencing era, showed dysregulation of cyclins-gene expression, as driver of the unique CLL features. Thus, our goal was to revisit the cell cycle in CLL, but focusing now in the NOTCH1 mutated subset (NOTCH1MUT), hypothesizing that biological differences versus wild type cases (NOTCH1WT) would explain the clinical ones, and exploiting potential differences with targeted molecules in vitro. Methods: From 2010 to 2019, presentation bone marrow aspirates or blood samples DNA was collected during the diagnostic workout from 378 CLL patients, all of them annotated by next generation sequencing. G 0/early-G 1 effectors gene expression was measured by RT-qPCR in negatively immunoselected circulating CLL cells. A siRNA approach was selected for silencing by electroporation 7 NOTCH1WT and 2 NOTCH1MUT cases. Cell cycle and apoptosis flow cytometry assays were performed on cultured fresh primary cells from n? NOTCH1MUT and 4 NOTCH1WT cases, before and after exposure to different concentrations of palbociclib, a CDK4/6 inhibitor. Results: We found that 37/378 (9.8%) of patients harbored a NOTCH1 mutation. NOTCH1MUTcases presented with higher lymphocyte counts [NOTCH1MUT 17.2 x10 9/L vs. NOTCH1WT 9.7 x10 9/L; p=0.042], trisomy 12 (35.1% vs. 11.4%; p<0.001) and a higher frequency of an unmutated IGHV status (70% vs. 21%; p<0.001). Of note, NOTCH1MUT patients had poorer responses to anti-CD20 based schemes than NOTCH1WT patients (35.7 vs. 69.8% complete response; p). We found that NOTCH1MUT cases showed a relevant increase of 38-fold change (FC) for CCND3, 27-FC for CDK4 and CCND2, 11-FC for CCND1 and 9-FC for CDK6 gene expression in negatively immunoselected circulating CLL cells at diagnosis. In addition, NOTCH1MUT cases displayed a statistically significant higher percentage of cells in the S phase than the wild type cases (21% vs. 1%, p=0.004). Though significance was not met, NOTCH1MUT cases showed a higher percentage of events within G 2-M (28% vs. 26 %). Next, we incorporated the flow cytometry assay to in vitro palbociclib treated CLL cells from 3 NOTCH1MUT and 4 NOTCH1WT cases. Five days after culture stimulation, cells were exposed to 38 and 76 μM (dose range for reaching maximum CLL cells sensitivity plateau) of the drug for 48 hours. As stated above, NOTCH1MUT cases were characterized by a much higher proportion of cells in S phase at baseline (21%), which was reduced in a dose dependent manner to an 8% and a 6% after exposure to palbociclib, respectively. The standard 48-72 hours drug assay may not be the most suitable for slow growth tumors as CLL and, in particular, for testing cell cycle inhibitors. Thus, we designed an assay for two cell cycles based on the average population doubling time of the primary cell culture experiments (0.6 in 72 hours), and using the mean steady state plasma concentration of palbociclib achieved clinically: 1 μM. After 120 hours, the baseline 15% of cells in S phase was reduced to an 0.64% after exposure to palbociclib 1 μM in NOTCH1MUT cases and a 1.8x-increase in the percentage dead cells was noted, compared with NOTCH1WT cases. Conclusions: Compared with NOTCH1WT CLL cases, we describe an overexpression of effectors of early phase in NOTCH1MUT. This profile made NOTCH1MU cells more suited to enter and traverse through the cell cycle and could explain, in part, the proliferative clinical-biological features of this subset of patients and opening a window for exploiting therapeutically these differences. Ours experiments in vitro with palbociclib sets the ground for the clinical research. Figure 1 Figure 1. Disclosures Jerez: BMS: Consultancy; Novartis: Consultancy; GILEAD: Research Funding.

Characterisation of a New Clinical Presentation of Chronic Lymphocytic Leukemia with Symptomatic Nasopharyngeal Mucosa Involvement

Patients with chronic lymphocytic leukemia (CLL) are prone to infectious complications, including Ears, Nose and Throat (ENT) infections due to the humoral immunodepression and/or to the immunosuppression related to the therapy. However, specific CLL infiltration in non-lymphoid regions of the head and neck causing ENT symptoms but unrelated to an infection is not well described. Extra-nodal localizations of CLL cells, including involvement of the mucosa of the rhinopharynx is uncommon and poorly reported. Here, we retrospectively analyzed the clinical, histopathologic and molecular features of 25 CLL patients with specific head and neck involvement. To date, this is the largest cohort reporting this new entity of CLL also named Nasal Associated Lymphoid Tissue (NALT) CLL. All patients had proven CLL prior to symptoms. Median time between ENT manifestation and CLL was 3 years [1-11 years]. Symptoms included chronic coughing (44%), antero-posterior nasal discharge (44%), nasal congestion (33%) and pharyngitidis (33%). ENT examination evidenced cervical lymphadenopathies in 68 % of cases, a granular aspect of the mucosa of the pharynx in 56%, enlarged tonsil (37%) or adenoids (37%). All patients underwent a biopsy of the mucosa of the nose or the throat. Histology and immunochemistry analysis demonstrated an infiltration of small lymphocytes CD20+, CD5+ and CD23+, consistent with a phenotype of CLL. The infiltration was diffuse in 50% of biopsies and perivascular in 38%. Patients with NALT-CLL had a poor prognosis: the majority was IGHV unmutated (n=18/25, 72%). Furthermore, they all required a treatment according to IWCLL criteria few time after the first ENT symptoms (median time 2 years), indicating that NALT-CLL is associated with a more progressive disease. To characterize the genetic background of NALT-CLL, we analyzed the cytogenetic data and NGS sequencing of 13 CLL-associated mutations from peripheral CLL cells. The karyotype was normal in only 2/24 cases (8%) and complex (>3 abnormalities) in 5/24 cases (20%). Half of the patients had a trisomy 12(12/24; 50%), while 13q, 17p and 11q deletions were found in 29%, 8% and 4% respectively. Eleven patients harbored one mutation (44%) while 9 patients had 2 to 5. Mutation of the NOTCH1 pathway was found in half of the cases (11/25 NOTCH1 and 2/25 FBXW7 mutated cases). TP53 and SF3B1 mutations occurred respectively in 20% (5/25 cases) and in 12% (3/25 cases). To gain insight into the molecular mechanism associated with involvement of the rhinopahrynx, we studied the expression of 70 genes related to cell migration and cell adhesion pathways using a qPCR array in the peripheral CLL cells of patients with NALT (n=4) compare to no NALT-CLL (n=4). Genes significantly up regulated with a fold change >2 included the chemokine receptors CCR7 (p=0.05) and CCR5 (p=0.04), the receptor involved in leukocyte trafficking CXCR3 (p=0.01) or the chemoattractant chemokine-like factor CKLF. By targeted qPCR, we confirmed the up regulation of CCR7 (p=0.002), CXCR3 (p=0.04) and CCR5 (p=0.03) in the cohort NALT-CLL patient (n=25) as compared to 20 age-matched CLL patients without head and neck symptoms (77% with unmutated IGHV, 30% with NOTCH1 mutation and 30% with trisomy 12). Up regulation of those targets was independent of the presence of NOTCH1/trisomy12 aberration or of the IGHV mutation status. These results suggest an increased migratory capacity of the leukemic CLL cells into the rhynopharynx mucosa related to a higher expression of these receptors involved in cell trafficking and migration. In line with these results, immunohistochemistry analysis of 5 patients with nasal involvement showed a strong staining of the CCR7 marker on the membrane of CLL cells infiltrating the mucosa. Interestingly, the staining of CCL21, the cognate ligand of CCR7, was positive in the vessels of the mucosa, suggesting that the recruitment and the transendothelial migration of CLL cells into the mucosa occur through a local secretion of CCL21 by the vessels. In summary, we report here a new presentation of CLL associated with symptomatic and specific ENT localization. CLL cells are predominantly IGHV unmutated, harbor NOTCH1 mutation and/or trisomy12 and show a higher expression of the chemokine receptors CCR7, CCR5 and CXCR3. We are currently studying the expression of those receptors by flow cytometry and the enhanced migratory capacity toward CCL21 through an in vitro chemotaxis assay. Disclosures Letestu: AbbVie: Research Funding, Speakers Bureau; Roche: Speakers Bureau; Janssen: Research Funding, Speakers Bureau. Cymbalista: Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; ASTRA ZENECA: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Lilly-LOXO: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Utilization of a Targeted Next Generation Sequencing Assay to Identify Copy Number Alterations in Chronic Lymphocytic Leukemia and Monoclonal B-Cell Lymphocytosis

Introduction: Chronic lymphocytic leukemia (CLL) is characterized by multiple copy number alterations (CNA) and mutations that are central to disease pathogenesis, prognosis, risk-stratification, and identification of response or resistance to therapies. Fluorescence in situ hybridization (FISH) is gold standard in the clinical laboratory for detecting prognostic CNAs in CLL (e.g. deletion 17p13 (del(17p), deletion 11q23 (del(11q), deletion 13q14 (del(13q), and trisomy 12). Most clinical FISH assays have high specificity and sensitivity, but the technique can detect a limited number of alterations per assay. Importantly, next-generation sequencing (NGS) techniques have become more readily available for clinical applications and are increasingly being used for screening not only mutations, but also copy number abnormalities in multiple genes and chromosomal regions of interest in hematologic malignancies. Here, we evaluated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) using a custom targeted NGS assay for detecting common prognostic chromosomal alterations in CLL and high-count monoclonal B-cell lymphocytosis (MBL), the precursor to CLL. Methods : We designed a SureSelect DNA targeted sequencing panel, covering all exons of 59 recurrently CLL mutated genes and additional amplicons across regions affected by clinically relevant CNAs. All CLL (N=534) and MBL (N=162) patients had pre-treatment peripheral blood mononuclear cells (PBMC) collected within two years of diagnosis. DNA was extracted in cases with purity >80% of CD5+/CD19+ cells. Clinical FISH data was available within 100 days of NGS in all untreated CLL and MBL cases. PatternCNV was used to detect clinically relevant CNAs in chromosomes 11, 12, 13 and 17. We performed a principal component analysis on the CNA calls, excluding chromosomes 11, 12, 13, and 17 to identify clusters of samples. Each cluster was then independently rerun with PatternCNV and the results from chromosomes 11, 12, 13, and 17 were extracted and further analyzed. We excluded samples with low tumor metrics identified by FISH (less than 20% of cells with del(17p), del(11q), trisomy 12 and del(13q)). Results: We sequenced a total of 696 patients of whom 162 were MBL and 534 were untreated CLL. The most commonly mutated genes were NOTCH1 (11.0%), TP53 (8.7%), SF3B1 (7.7%), ATM (4.1%), and CHD2 (3.8%). Based on CNA analyses from the NGS data, we identified 59 (9.1%) individuals with del(17p), 88 (13.4%) individuals with del(11q), 128 (20.0%) individuals with trisomy 12, and 329 (53.0%) individuals with del(13q). All 696 individuals had FISH panels conducted, with 39 (5.6%) individuals with del(17p), 68 (9.8%) individuals with (11q), 119 (17.1%) with trisomy 12, and 295 (42.4%) with del(13q). When we compared our CNA analyses with the FISH data, we found high concordance 95.0% for del(17p), 92.7% del(11p), 94.3% for trisomy 12, and 88.2% for del(13q). For del(17p) we found a sensitivity of 93.9%, specificity of 95.4%, PPV of 52.5%, and NPV of 99.7%. Del(11q) revealed a sensitivity of 88.1%, specificity of 94.0%, PPV of 59.1%, and NPV 98.8%. We found a sensitivity of 93.8%, specificity of 95.6%, PPV 82.0%, and NPV of 98.6% for trisomy 12 and for del(13q) we found a sensitivity of 92.6%, specificity of 90.9%, PPV of 91.7%, and NPV of 93.8%. We found lower PPVs in del(17p) and del(11q) likely due to lower prevalence of these chromosomal abnormalities. Conclusion: Here we show a high sensitivity, specificity, and NPV when comparing targeted sequencing with FISH. FISH panel testing is widely used in clinical practice to characterize highly prognostic chromosomal abnormalities in CLL. Comprehensive genetic profiling with NGS has become increasingly important in the work up of hematologic malignancies and provides additional prognostic and predictive information, including clinically relevant mutations such as TP53, SF3B1, and NOTCH1, tumor mutation load and mutations associated with resistance to chemo-immunotherapy and targeted therapies, such as BTK or BCL2 inhibitors, that FISH cannot offer. We show that NGS can infer clinically relevant CNA in cases without FISH testing while also providing additional clinically relevant information. Figure 1 Figure 1. Disclosures Cerhan: Regeneron Genetics Center: Other: Research Collaboration; Celgene/BMS: Other: Connect Lymphoma Scientific Steering Committee, Research Funding; NanoString: Research Funding; Genentech: Research Funding. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: Genentech: Research Funding; MEI Pharma: Research Funding; Sunesis: Research Funding; Acerta Pharma: Research Funding; 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; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Behring: Membership on an entity's Board of Directors or advisory committees.

Activity of Limited-Schedule Bendamustine and Methylprednisolone in Chronic Lymphocytic Leukemia

Bendamustine was approved by the United States Food and Drug Administration in 2008 for the treatment of Chronic Lymphocytic Leukemia (CLL). Corticosteroids have been administered in regimens with chemotherapy for CLL for more than 40 years. Despite this, the combination of Bendamustine and Methylprednisolone has not been explored in CLL. We describe our experience with Bendamustine 100 mg/m2 and Methylprednisolone 125 mg IV on days 1 and 2 and Peg-filgastrim 6 mg subcutaneously on day 3 every 28 days for 3-4 cycles in 12 patients with CLL. Cycles have been limited to attempt to prevent cumulative marrow toxicity due to Bendamustine. Patient characteristics: 7 males/5 females, median age-58, Binet stage B (8), C (2), A (2); ZAP70/CD38+ (5), Trisomy 12 (3), 13q- (2), normal cytogenetics (2); median ECOG status 1, no prior therapy in 8. Median number of cycles received = 3 (two received 4 cycles). Most common toxicities: nausea/emesis (5), fever/flu-like symptoms (4), arthralgia/myalgia (4). There have been no treatment-related deaths. One patient was briefly hospitalized for pneumonia. Complete responses have been seen in 6 patients (median duration = 50.7 months; range: 12.1+ to 87.3 months). Partial responses have been seen in 4 patients (median duration = 17.3+ months; range: 3.3 to 26.9+ months). The combination of Bendamustine and Methylprednisolone given in a limited schedule is well-tolerated and is active in CLL. Disclosures No relevant conflicts of interest to declare.

Accurate Prediction of Chromosome-Level CNVs from Targeted NGS

Background: Aneuploidy and large-scale Copy Number Variations (CNVs) are prominent features of cancer cells. While Fluorescence in situ hybridization (FISH) and conventional cytogenetics (CC) are the gold standard for detecting aneuploidy and CNVs, NGS-based assays are currently used for high-resolution detection of copy number alterations assessing the whole genome. However, although an increasing number of NGS-based tools have been developed for detecting aneuploidy or CNVs from whole genome or exome sequencing data, only a limited number of options are available for targeted gene panels. Despite mechanisms provided to establish normal profiles for a specific panel, the accuracy of these tools at the chromosome level suffer when only a small number of regions are targeted on each chromosome. Here we leveraged on a custom amplicon based NGS assay designed to detect somatic alterations (SNVs and indels) in 297 hematological cancer relevant genes, previously validated in our clinical laboratory. We introduce a simple approach to accurately predict chromosome-level CNVs such as monosomy and trisomy for a targeted gene panel, commonly used in a clinical setting. Methods: Mutation profiles, including SNVs, INDELs, and structural changes, were interrogated with an in-house bioinformatics pipeline that utilized PureCN and CNVkit algorithms to detect structural changes. The first step consists of finding optimal panel-specific decision thresholds for gains and losses at the gene level. This step was performed using an independent set of 1,314 clinical samples sequenced with the NeoType® Heme assay developed by NeoGenomics Laboratories, Inc. for which at least one FISH test was performed in addition to the sequencing. Three genes (ATM, TP53, and NF1) were used to find optimal decision thresholds based on the FISH result for these markers. These thresholds are used afterward to predict a gain or a loss for any other gene in the panel. The second step consists of predicting the chromosome-level gain or loss based on the individual predictions at the gene level by simply observing the frequency of targeted genes on the corresponding chromosome predicted as either gained or lost by the first step approach. The 19, 7, and 18 targeted genes in the NGS panel (Table 1) were respectively used to predict monosomy 7, trisomy 8, and trisomy 12 in a second set of over 7,000 clinical samples with known ploidy for chromosomes with clinically relevant ploidy abnormalities in hematological malignancies. Results: Evaluation of the first stage gene-level CNV prediction on 1,314 clinical samples shows a concordance rate of 97.95% between NGS and FISH results on ATM, TP53, and NF1. When we evaluated the second stage chromosome-level CNV prediction in clinical samples sequenced using the same targeted panel and assessed by FISH for chromosome-level variation on chromosomes 7, 8 and 12 (Table 1), a heatmap of the predicted Log 2 ratios for each sample and targeted gene from the first step shows a clear distinctive signal between aneuploidy and diploid samples (Figure 1). At the chromosome level, the concordance rate between the final prediction and the FISH results is consistently observed above 93% (Table 2). Roughly 50% of the 12, 78, and 40 discordant calls for monosomy 7, trisomy 8, and trisomy 12, respectively captured by FISH but not by NGS can be explained by low tumor content (less than 20%) in the tested samples. The concordance rate between NGS and FISH is consistently observed above 96% when leaving these samples aside. Note that results in Table 2 are obtained using all samples to decide the optimal decision threshold for the chromosome-level prediction, but are found identical when using a leave-one-out evaluation procedure, and nearly identical when using a repeated cross-validation procedure. Conclusion: This study demonstrates that chromosome-level CNVs can be accurately predicted in hematologic malignancies even when the number of targeted genes on a given chromosome is low. Despite the simplicity of the approach, the two stages bioinformatics pipeline based on an ensemble method allowed us to gain between 8% and 46% accuracy compared to relying only on the prediction of a single tool like PureCN. Samples with low tumor content remain, however, a difficult case to tackle with bulk NGS as it is difficult to distinguish a CNV from the natural variability of the sequencing coverage. Figure 1 Figure 1. Disclosures Nam: NeoGenomics Laboratories, Inc.: Current Employment. Magnan: NeoGenomics Laboratories, Inc.: Current Employment. Lopez-Diaz: NeoGenomics Laboratories, Inc.: Current Employment. Bender: NeoGenomics Laboratories, Inc.: Current Employment. Agersborg: NeoGenomics Laboratories, Inc.: Current Employment. Jung: NeoGenomics Laboratories, Inc.: Current Employment. Funari: NeoGenomics Laboratories, Inc.: Current Employment.

Proteomic Profiling Based Classification of CLL Provides Prognostication for Modern Therapy and Identifies Novel Therapeutic Targets

The availability of targeted therapy and improved molecular characterization of Chronic Lymphocytic Leukemia (CLL) require a re-evaluation of treatment paradigms. As CLL heterogeneity is dependent on molecular and environmental factors, there is a need to create a new classification based on the integration of several factors. Here, we accomplish this goal by identifying CLL signatures using Reverse Phase Protein Array. Protein expression for 384 total and post translationally modified proteins was assessed in 871 CLL and Mature Small B Cell Leukemia (MSBL: HCL, HCLV, LGL-T, MCL, MZL, PLL, Richter's, T-Cell PLL) patients and was integrated with clinical data to identify strategies for improving diagnostics and therapy, making this the largest CLL proteomics study to date. Proteins were categorized into 40 protein functional groups (PFGs) based on literature and intra-dataset protein correlations and patients clustered based on PFG expression patterns into 6 recurrent protein expression signatures (PES) (Figure 1A). Individual protein expression (58/384 proteins), PFG expression (32/40) and overall PES were all highly prognostic of survival (OS) and time to first or second treatment (TTFT, TTST) (Figures 1B-C). The adhesion, apoptosis-occurring, apoptosis-regulating, heat shock, histone1 (marks), histone 2 (modifiers) and the STP-regulation PFGs were prognostic for all 3 outcome measures. Notably SG-A contained most of the MSBL and 15/16 cases of hairy cell leukemia, but the CLL cases within this SG fared very poorly. For OS, groups A and C had markedly inferior survival (P<0.0001) (10.3 and 20.3 median years) relative to the other 4 groups, which were statistically similar to each other. First treatment occurred sooner for Groups A and C (5.8 and 5.23 median years). Additionally, the TTST was also inferior for Group A (median 3.5 years). There were significant differences in age, hemoglobin, platelets, % BM and PB lymphocytes and β2M between the SG, but not for race (p = 0.84) or gender (p = 0.72). , Historically adverse cytogenetic aberrations del 11q and del17p events (23% overall) were less common in SG A, B, D and E (15, 14, 16, 17%) and overrepresented in SG C (32%), while historically favorable 13q changes were seen across all groups as was Trisomy 12 (14% overall), although SGs A and E were enriched (25%, 22%) while SG-F was low (5%) for Trisomy 12. SG membership superseded other traditional prognostic factors (Rai Staging, IGHV Status) and were prognostic for modern (BTK inhibition) and older CLL therapies. SGs A and C responded poorly to chemotherapy regimens compared to the other groups, whereas all groups responded well to BTK inhibitors except for SG-A. SGs and PFGs membership provided novel drug targets (see our other abstracts on TP53BP1 and ASNS) and defined optimal candidates for Watch and Wait (WaW) vs. early intervention. A model based on the accumulation of irregularities in ANXA1, TFRC, and SMAD2.p245 expression, optimally predicted TTFT overall and in early stage CLL patients. Patients with < 1 negative level of either of the 3 proteins, have a median TTFT of 14.59 years, whereas patients having 2-3 have a median of 5-6.27 years (P<0.0001). CHEK1.pS345, GAB2, IGFBP2, S100A4, WEE1.pS642, and ZAP70 were universally overexpressed by all SGs, suggesting them as ideal targets for inhibition. Collectively proteomics demonstrates promise for improving classification, therapy strategy determination, and identifying novel therapeutic targets. Figure 1 Figure 1. Disclosures Ferrajoli: Janssen: Other: Advisory Board ; AstraZeneca: Other: Advisory Board, Research Funding; BeiGene: Other: Advisory Board, Research Funding. Thompson: Genentech: Other: Institution: Advisory/Consultancy, Honoraria, Research Grant/Funding; Amgen: Other: Institution: Honoraria, Research Grant/Funding; Adaptive Biotechnologies: Other: Institution: Advisory/Consultancy, Honoraria, Research Grant/Funding, Expert Testimony; Pharmacyclics: Other: Institution: Advisory/Consultancy, Honoraria, Research Grant/Funding; Janssen: Consultancy, Honoraria; Gilead: Other: Institution: Advisory/Consultancy, Honoraria; AbbVie: Other: Institution: Advisory/Consultancy, Honoraria, Research Grant/Funding. Burger: Pharmacyclics LLC: Consultancy, Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; Beigene: Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; Gilead: Consultancy, Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel/Accommodations/Expenses, Speakers Bureau; AstraZeneca: Consultancy; Janssen: Consultancy, Other: Travel/Accommodations/Expenses, Speakers Bureau. Wierda: Xencor: Research Funding; Karyopharm: Research Funding; Gilead Sciences: Research Funding; Acerta Pharma Inc.: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; AstraZeneca: Research Funding; Juno Therapeutics: Research Funding; KITE Pharma: Research Funding; Sunesis: Research Funding; Miragen: Research Funding; Oncternal Therapeutics, Inc.: Research Funding; Cyclacel: Research Funding; Loxo Oncology, Inc.: Research Funding; Janssen: Research Funding; Genentech: Research Funding; GSK/Novartis: Research Funding; Genzyme Corporation: Consultancy; AbbVie: Research Funding.

Cause and effect relationship between application of deuterium-depleted water and improvement of chronic lymphocytic leukemia

The anticancer effect of deuterium depletion has been proved in various types of cancer in combination with conventional therapies. Here we report a case study of a patient diagnosed in 2006 with chronic lymphocytic leukemia (CLL). His stage at diagnosis did not require the immediate conventional therapy and so he started consuming deuterium-depleted water (DDW). Significant changes occurred already during the first three months; WBC count dropped from 16x103 cells/µL to 7.7x103 cells/µL, to the normal range and the cervical lymph nodes regressed. He continued drinking DDW uninterruptedly in the first three years (1,231 days) and periodically in the subsequent 11 years. PLT count significantly increased from 108x103 /µL to 232x103 /µL after two years’ DDW-consumption. CD5+/CD19+ percentage showed significant decrease from 69% to 4% and the rate of chromosome trisomy 12 in interphase blood cells was reduced from 40% to 7% in the first three years. The study confirms that deuterium depletion as a single treatment is effective and delays the use of conventional chemotherapy.