Abstract Lung cancer is the number one cause of cancer-related deaths in both men and women with a median survival time of 8-10 months’ post treatment. Non Small Cell Lung Cancer (NSCLC) accounts for 80% of the lung cancers and the treatment plan is determined based on the active mutations (EGFR, ALK/ROS, and KRAS) present in the tumor. Patients bearing EGFR mutation initially respond to targeted tyrosine kinase inhibitor (TKI) therapy and after 10-14 months of treatment acquire TKI resistance. Consequently, the median PFS for NSCLC patients with EGFR mutation is only 9.5 months. The reason for this acquired drug resistance is not yet fully understood. Recent studies have reported oncogenes such as AXL could be responsible for TKI resistance. Therefore, understanding the mechanism of drug resistance is key in developing a solution to overcome the problem. For this study, we first examined the resistance mechanism and developed a biodegradable targeted nanoparticle based solution to systematically investigate the role of AXL in resistant NSCLC cell lines. In this study we (1) downregulated AXL using siRNA and separately (2) knocked out the AXL gene using crRNA (CRISPR) and treated with TKI. Our results show that AXL is responsible for activation of several EMT related proteins and upregulation of mTOR pathway. We believe the upregulation of these proteins is essential for cancer cells to switch pathways for proliferation and regulating miRNAs linked to mutations. Our results further confirm that AXL is responsible for regulating MMP-2 that is associated with cell invasion. Based on data from multiple cell lines such as H820 and A549, we demonstrate that AXL upregulation is responsible for resistance independent of EGFR activating mutations. The elucidated pathway for drug resistance was further confirmed in cell lines generated by knocking-out the AXL gene. Interpretation for AXL signaling and drug resensitization was confirmed by Western blotting, Zymography, Invasion & Migration assay, Apoptosis assay and MTT toxicity assay. We performed mRNA and miRNA analysis using qRT-PCR to understand gene expression post treatment. During the process of our investigation, we found that NSCLC cells undergo further survival cross talk with other biomarkers. Indeed, we report the first experimental evidence of a survival cross talk between AXL and FN14, a wound healing gene, that enhance cell survival post treatment. Down regulation of both AXL and FN14 dramatically reduced the IC50 of TKI. Based on the mechanism, we designed a gelatin nanoparticle that can carry both AXL and FN14 to deliver it in the tumor cell. The nanoparticle is targeted to tumor using EGFR-antibody and releases the silencing RNA within cytoplasm. We further demonstrated that dual-inhibition of AXL and FN14 in A549 mice xenografts showed tumor reduction compared to controls. In conclusion, inhibition of AXL and FN14 can maximize therapeutic response of TKI, wherein AXL is upregulated before or during drug treatment. Citation Format: Dhananjay Suresh, Ajit Zambre, Soumavo Mukherjee, Shreya Ghoshdastidar, Jennifer L. Schnabel, Sarah Chapman, W. Matthew Leevy, Anandhi Upendran, Raghuraman Kannan. Combined AXL/FN14 inhibition sensitize drug-resistant NSCLC in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4171. doi:10.1158/1538-7445.AM2017-4171

OBJECTIVES Certain laboratory tests are critical for patient care and must be communicated rapidly. Professional societies have developed guidelines for reporting of "critical values" and significant or unexpected surgical pathology findings. Urgent diagnoses requiring rapid reporting include new or unexpected diagnoses of malignancy. The University of Missouri established a "cancer-tracking" protocol in which all diagnoses of malignancy require acknowledgment by the responsible clinician. METHODS We reviewed 5 months of compliance with the cancer-tracking protocol. The protocol requires the Department of Pathology to send a letter to the responsible clinician requesting acknowledgment of the report. In total, 1,155 confirmation requests were sent to the physicians named on the request form. RESULTS Following the first letter, 692 acknowledgments were received, and 356 acknowledgments followed the second letter. In 107 (9%) cases, no response was received. CONCLUSIONS Confirmation that the physician caring for a patient receives a pathology diagnosis is important for patient management and quality assurance. While the surgical pathology report was placed in the chart, it was impossible to confirm that the responsible physician had reviewed the report in 9% of cases. Techniques for communication and confirmation of transmission of anatomic pathology diagnoses need to be developed.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation. This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

We conducted a genome-wide DNA methylation analysis in CD19 (+) B-cells from chronic lymphocytic leukemia (CLL) patients and normal control samples using reduced representation bisulfite sequencing (RRBS). The methylation status of 1.8-2.3 million CpGs in the CLL genome was determined; about 45% of these CpGs were located in more than 23,000 CpG islands (CGIs). While global CpG methylation was similar between CLL and normal B-cells, 1764 gene promoters were identified as being differentially methylated in at least one CLL sample when compared with normal B-cell samples. Nineteen percent of the differentially methylated genes were involved in transcriptional regulation. Aberrant hypermethylation was found in all HOX gene clusters and a significant number of WNT signaling pathway genes. Hypomethylation occurred more frequently in the gene body including introns, exons, and 3'-UTRs in CLL. The NFATc1 P2 promoter and first intron was found to be hypomethylated and correlated with upregulation of both NFATc1 RNA and protein expression levels in CLL suggesting that an epigenetic mechanism is involved in the constitutive activation of NFAT activity in CLL cells. This comprehensive DNA methylation analysis will further our understanding of the epigenetic contribution to cellular dysfunction in CLL.

Abstract Chronic lymphocytic leukemia (CLL) is the most common leukemia of adults in the western world. The clinical course of CLL is highly variable. Some patients are able to live years with no need for treatment; whereas others progress rapidly requiring therapy within a short time after diagnosis. The identification and validation of prognostic molecular markers (including surface markers, cytogenetic abnormalities, and IGHV mutational status) have resulted in refinements in the approach to the management of these patients. Further discovery of biologically relevant factors that influence the heterogeneity and progression of CLL will not only promote our understanding of the disease process, but also allow us to identify rational therapeutic approaches. In this study, we conducted genome-wide DNA methylation analyses in purified CD19+ B-cells from 11 CLL patients with a range of CD38 expression using reduced representation bisulfite sequencing (RRBS). Using one lane of the Illumina sequencing data, we were able to consistently determine the methylation status of approximately 1.8 million CpGs; 45% of these CpGs are located in more than 23,000 CpG islands (CGIs), accounting for more than 80% of all annotated CGIs across the genome. We have identified a large number of CGIs that were differentially methylated between CLL cells and normal CD19+ B-cells. The promoter hypermethylation patterns of many genes previously reported in CLL, such as FOXD3, GRM7, DLEU7 etc, were determined in the 11 CLL samples. In addition, we have observed aberrant DNA methylation changes in all 4 HOX gene clusters in CLL. For instance, HOXD8, HOXD9, and HOXD11 were hypermethylated in 11 out of 11 CLL samples. In addition, we also identified a significant number of DNA methylation alterations in the WNT signaling pathway genes. One-way ANOVA analysis of DNA methylation profiles of 764,984 CpGs shared by all CLL samples identified 570 CpGs that were differentially methylated (p<0.001) between CD38high and CD38low group (with >20% CLL cells expressing CD38 as CD38high group). The cluster analysis of the 570 CpGs reveals that it is possible to separate these CLL samples into two distinct groups based on the DNA methylation profiles. The study further confirms our early findings that CLL is affected by CpG island methylation in some genes that segregate with CD38 expression levels, while most others show similar methylation patterns across all levels. The aberrant promoter hypermethylation in certain functional gene groups and pathway-associated genes that are known to be deregulated in CLL provides additional insights into the CLL methylome and epigenetic contribution to cellular dysfunction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4791. doi:10.1158/1538-7445.AM2011-4791

Background: Follicular lymphoma (FL) is a form of non-Hodgkin's lymphoma (NHL) that arises from germinal center (GC) B-cells. Despite the significant advances in immunotherapy, FL is still not curable. Beyond transcriptional profiling and genomics datasets, there currently is no epigenome-scale dataset or integrative biology approach that can adequately model this disease and therefore identify novel mechanisms and targets for successful prevention and treatment of FL.Methodology/Principal Findings: We performed methylation-enriched genome-wide bisulfite sequencing of FL cells and normal CD19(+) B-cells using 454 sequencing technology. The methylated DNA fragments were enriched with methyl-binding proteins, treated with bisulfite, and sequenced using the Roche-454 GS FLX sequencer. The total number of bases covered in the human genome was 18.2 and 49.3 million including 726,003 and 1.3 million CpGs in FL and CD19(+) B-cells, respectively. 11,971 and 7,882 methylated regions of interest (MRIs) were identified respectively. The genome-wide distribution of these MRIs displayed significant differences between FL and normal B-cells. A reverse trend in the distribution of MRIs between the promoter and the gene body was observed in FL and CD19+ B-cells. The MRIs identified in FL cells also correlated well with transcriptomic data and ChIP-on-Chip analyses of genome-wide histone modifications such as tri-methyl-H3K27, and tri-methyl-H3K4, indicating a concerted epigenetic alteration in FL cells.Conclusions/Significance: This study is the first to provide a large scale and comprehensive analysis of the DNA methylation sequence composition and distribution in the FL epigenome. These integrated approaches have led to the discovery of novel and frequent targets of aberrant epigenetic alterations. The genome-wide bisulfite sequencing approach developed here can be a useful tool for profiling DNA methylation in clinical samples.

High‐throughput microarray technologies were used to study DNA methylation accompanied by transcriptional changes in follicular lymphoma (FL). Using Methylated CpG Island Amplification with Microarrays to study CpG Island DNA methylation in FL, we discovered widespread hypermethylation of homeobox genes and previously identified targets of polycomb repressive complex 2 (PRC2) in cell lines and primary tumors, but not in benign follicular hyperplasia (BFH). DNA methylation for HOXA11 , HOXD10 , HOXB7 , HOXC12 , PAX6 , LHX9 , SFMBT2 , EN2 , and PAX7 was independently validated in the RL cell line and HOXA11 , HOXD10 , PAX6 , and EN2 in primary tumors. Combined Bisulfite Restriction Analysis (COBRA) also established DNA methylation for the previously identified PRC2 targets DCC , DES , GAD2 , AQP5 , GPR61 , GRIA4 , GJD2 , and AMPH in FL but not in BFH. Gene expression analyses revealed 411 genes that were hypermethylated and transcriptionally repressed in RL, 74% of which were reactivated by the demethylating agent 5‐aza‐2′‐deoxycytidine (5‐azaD) plus or minus the histone deacetylase inhibitor trichostatin A (TSA). Forty genes were also downregulated in primary FL. Our results suggest that extensive hypermethylation in promoters of polycomb target genes is a characteristic of FL and that loss of expression of certain SUZ12 target genes could be functionally relevant for lymphomagenesis. © 2009 Wiley‐Liss, Inc.