Citations

Abstract Glioblastoma (GBM) is the most common primary tumor of the CNS and carries a dismal prognosis. The aggressive invasion of GBM cells into the surrounding normal brain makes complete resection impossible, significantly increases resistance to the standard therapy regimen, and virtually assures tumor recurrence. Median survival for newly diagnosed GBM is 14.6 months and declines to 8 months for patients with recurrent GBM. New therapeutic strategies that target the molecular drivers of invasion are required for improved clinical outcome. We have demonstrated that TROY (TNFRSF19), a member of the TNFR super-family, plays an important role in GBM invasion and resistance. TROY expression increases with glial tumor grade and inversely correlates with patient survival. TROY stimulates GBM cell invasion and increases resistance to temozolomide (TMZ) and radiation treatment. Conversely, knockdown of TROY expression inhibits GBM cell invasion, increases sensitivity to temozolomide, and prolongs survival in an intracranial xenograft model. Propentofylline (PPF), an atypical synthetic methylxanthine compound, has been extensively studied in Phase II and Phase III clinical trials for Alzheimer's disease and vascular dementia where it has demonstrated blood-brain permeability and minimal adverse side effects. In this study, we demonstrated that PPF decreases TROY protein expression in glioma cells, and subsequently suppress activation downstream signaling effectors including AKT, NF-κB, and Rac1. PPF treatment of glioma cells also suppressed glioma cell migration and invasion, demonstrated by transwell migration assay and reduced membrane ruffling. Finally, PPF treatment increased vulnerability of glioma cells to Temozolomide (TMZ) and radiation. In summary, this study demonstrates that PPF provides a pharmacologic approach to target TROY to inhibit glioma cell invasion and reduce therapeutic resistance to TMZ and radiation. Citation Format: Harshil D. Dhruv, Serdar Tuncali, Alison Roos, Patrick Tomboc, Nathan Jameson, Ashley Chavez, Joseph Loftus, Michael E. Berens, Nhan L. Tran. Propentofylline inhibits TROY/TNFRSF19 signaling to enhance therapeutic efficacy in invasive glioblastoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5366. doi:10.1158/1538-7445.AM2015-5366

Abstract Introduction Recent advances in stereotactic radiosurgery (SRS) have improved clinical outcomes in patients with limited intracranial metastases. As a result of this high-dose radiation delivery to a focal area within the brain, tissue structural and functional changes within the treatment field are often difficult to distinguish from local tumor recurrence based on conventional imaging techniques. Here, we demonstrate that elevation of circulating cells of myeloid origin, the key regulators of inflammation and immune responses, can serve as a potential surrogate marker to differentiate radiation-induced tissues changes from local tumor recurrence. Materials and Methods We retrospectively identified 96 patients who were initially treated with Gamma-knife radiosurgery at MD Anderson Cancer Center from 2009-2013 for brain metastases, and subsequently underwent surgical resection of previous irradiated lesions after presenting with clinical symptoms and radiographic findings suggestive of tumor progression. All surgical specimens were centrally reviewed by neuropathology. Pre-operative circulating blood cell profiles were analyzed for monocytic, granulocytic and lymphocytic sub-populations. Results A total of 11 and 85 patients who underwent surgical resection after prior SRS for brain metastasis were pathologically confirmed to have radiation-induced changes or tumor recurrence, respectively. Among patients with confirmed recurrence vs. those with radiation-induced changes, the number of circulating immature granulocytes (mean = 0.093 × 103ul−1 vs 0.032 × 103ul−1, p = 0.075) and monocytes (mean = 0.632 × 103ul−1 vs 0.401 × 103ul−1, p = 0.007) were elevated when compared to patients with radiation-induced changes (combined: mean = 0.71 × 103ul−1 vs 0.43 × 103ul−1, p = 0.002). No statistically significant differences were observed among mature granulocytic or lymphocytic populations between the two groups. Receiver operating characteristic analysis showed significant discriminative utility of pre-operative myeloid cell count when compared to reference line of no discrimination (AUC = 0.774, p = 0.003). The cut-point for myeloid cell population was found be > = 0.5 × 103ul−1, which corresponds to a detection sensitivity of 73.3% and specificity of 72.7% for tumor recurrence. Conclusion Bone marrow cells of myeloid origin play important roles in mediate acute inflammatory reactions and in regulating adaptive immunity. We showed here that certain primitive cells of myeloid lineage may potentially be used to aid the differentiation of tumor recurrence from radiation-induced changes in patients who had SRS for brain metastasis. The identification of potential circulating biomarkers that can complement traditional imaging to distinguish the two distinct pathological entities has significant implications in the management of these patients. Note: This abstract was not presented at the meeting. Citation Format: Wen Jiang, Yvo Rodriguez, Nicolas S. Boehling, Sujit S. Prabhu, Betty Y.S. Kim, Patrick Hwu, Erik P. Sulman, Paul D. Brown, Jing Li. Circulating myeloid precursor profile as potential marker to differentiate radiation changes from tumor recurrence after brain stereotactic radiosurgery. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 532. doi:10.1158/1538-7445.AM2015-532

Abstract Background: Protein kinase Cβ (PKCβ) is critical for B-cell signaling and survival and overexpression of PKCβ in DLBCL is associated with inferior survival. ENZA, an oral serine/threonine kinase inhibitor, targets PKCβ. Here we report immunohistochemical (IHC) and fluorescence in-situ hybridization (FISH) correlative analyses. Methods: PRELUDE (NCT00332202) was a phase III, double-blind maintenance study of 758 pts with DLBCL who were randomized 2:1 to ENZA 500 mg daily (1125-mg loading dose) (n = 504) or PBO (n = 254), respectively, following CR to induction therapy with R-CHOP. The primary endpoint was disease-free survival (DFS). Overall survival (OS) and assessment of biomarkers specific to ENZA and DLBCL were secondary endpoints. Pre-treatment formalin-fixed, paraffin-embedded samples were assessed via IHC staining in a blinded manner (Eric Hsi) for cell of origin (COO) using Hans’ algorithm. In addition, IHC was performed for c-MYC, BCL2, BCL6, FOXP1, and MUM1 (10% increments/% tumor cells stained), markers relevant to ENZA, including PKCβ2. FISH was performed to identify translocations involving c-MYC, BCL2, and BCL6. Cox regression was used to determine statistical associations between efficacy outcomes and dichotomized markers, adjusting for treatment and additional baseline covariates. All tests of association were conducted at a 2-sided α = 0.05. Results: There was no difference in 4-year DFS (70% vs 71%) or OS (81% vs 82%) between ENZA and PBO arms, respectively. A total of 243 (32%) pts had ≥1 evaluable sample available for IHC and FISH. There was no difference in outcome for pts based on COO (GCB vs non-GCB). Independent of treatment, significant associations were observed for BCL2 (pre-specified cutpoint 20%) and MUM1 (cutpoint 30%) with OS, and FOXP1 (cutpoint 80%) by IHC with DFS (HR [95% CI]: 2.19 [1.01-4.73]), p = 0.031; 1.97 [1.04-3.71], p = 0.032; 1.74 [1.04-2.90], p = 0.032, respectively). Associations were not significant for other IHC markers, including c-MYC and BCL6. Low PKCβ2 (<50% expression) had numerically better (but not significant) DFS/OS vs high PKCβ2. Dual translocation lymphoma by FISH was identified in two pts (1.2%) each for c-MYC/BCL2 and c-MYC/BCL6, and one pt (0.6%) had a triple hit involving c-MYC/BCL2/BCL6. Conclusions: No difference in outcomes between treatment arms was observed for the trial. Independent of treatment, COO was not prognostic of outcomes. Pts with low PKCβ2 expression had numerically better DFS/OS compared with high PKCβ2 expression. Significant treatment-independent associations were observed for BCL2 and MUM1 with OS and for FOXP1 with DFS (low IHC expression corresponded to better outcomes). The small number of double hit and triple hit lymphomas seen in the study may reflect the enrollment of CR patients with more favorable biology. Citation Format: Eric D. Hsi, Kerry J. Savage, Sonali M. Smith, Fritz Offner, Scott P. Myrand, Thomas M. Habermann, Donald E. Thornton, Boris K. Lin, Tuan S. Nguyen, Oday Hamid, Michael Crump. Correlative results from PRELUDE, a phase III study of enzastaurin (ENZA) vs placebo (PBO) in patients (pts) with high-risk diffuse large B-cell lymphoma (DLBCL) following a response to R-CHOP therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5303. doi:10.1158/1538-7445.AM2015-5303

Abstract Introduction. CLL has a highly heterogeneous clinical course. In fact, while some patients experience a very aggressive disease, others may present with an indolent form, and eventually die of “other” causes. Here we report the findings of a prospective cohort study evaluating the comorbidities and causes of death in newly diagnosed patients with CLL. Methods. Clinical information was collected from 1174 patients with newly diagnosed CLL (within 9 months of diagnosis), who consented to participate in a prospectively observational trial with standardized longitudinal follow-up between 01/2002 and 11/2014. All comorbidities were diagnosed before presentation. Results. Baseline characteristics are shown in the Table. Co-existent health conditions included: cardiac (28% of patients), respiratory (18%), endocrinological (14%), rheumatologic (42%), gastrointestinal (33%), genitourinary (35%), and psychiatric diseases (17%), diabetes (10%), hyperlipidemia (41%), other cancers (20%), stroke (3%), hypertension (40%), venous thromboembolism (3%), substance abuse (5%), sexually-transmitted infections (3%), and obesity (32%). After a median follow up of 4 years, 197 patients died and the cause of death could be accurately determined in 135 patients. Among these, death was due to CLL progression in 69 (51%) patients, infections in absence of CLL progression in 7 (5%), other cancers in 29 (21%), and other comorbid health conditions in 30 (22%). CLL patients (N = 1174)Number (%), median [range]Age (years)63 [23-89]Males791 (67)Females383 (33)Rai stage 0604 (51)I-II512 (44)III-IV54 (4)missing4 (1)B2M < 4 mg/dL717 (61)> 4 mg/dL97 (8)missing360 (31)CD49d negative638 (54)positive277 (24)missing259 (22)CD38 < 30%780 (66)> 30%332 (28)missing62 (6)ZAP70 negative650 (55)positive380 (32)missing135 (13)IGHV mutated506 (43)unmutated394 (34)missing274 (24)FISH del13q395 (34)negative173 (14)+12276 (23)del11q90 (8)del17p50 (4)other2 (1)missing188 (16) Conclusions. Despite the advanced age at CLL diagnosis, the frequent presence of comorbidities, and the indolent nature of the disease, CLL progression is the ultimate cause of death in 51% of newly diagnosed CLL patients, with an additional 26% dying of causes potentially related to CLL, such as infections and second cancers. The influence of comorbidities and other clinical factors at diagnosis (e.g. smoking, Charlson Comorbidity Index) on survival and ultimate cause of death are being abstracted, and will be presented at the meeting. Citation Format: Paolo Strati, Kari Chaffe, Sara Achenbach, Timothy Call, Neil Kay, James Cerhan, Susan Slager, Tait Shanafelt. Comorbidity and cause of death in patients with chronic lymphocytic leukemia (CLL). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5267. doi:10.1158/1538-7445.AM2015-5267

Abstract Androgen deprivation therapy (ADT) for hormone-sensitive prostate cancer (HSPC) and chemotherapy for castration-resistant prostate cancer (CRPC) are considered standard of care. However, there are no clinical tests or factors that can reliably predict treatment responses for the advanced prostate cancer patients. Examination of tumor component in circulation, also known as liquid biopsy, has shown promise for predicting treatment outcomes and survival. To capture the tumor-derived genomic alterations and decrease the surrogacy of using PSA measurements as is currently performed, we propose a Plasma Genomic Abnormality (PGA) score and Treatment Efficacy (TEff) index based on copy number variations as estimated by cell free DNA (cfDNA) in plasma. We first performed whole genome and targeted sequencing (NimbleGen comprehensive cancer panel) to examine plasma cfDNA for tumor-associated genetic and genomic aberrations in 10 HSPC patients receiving ADT and 10 CRPC patients receiving chemotherapy. For each patient, we tested plasma from two time points: pre-treatment and 4 months post-treatment. We then calculated the PGA score by summing the most significant genomic abnormalities with higher PGA score indicating greater tumor DNA content in cfDNA. TEff index was derived from comparison of PGA score difference between treatments with higher TEff index reflecting a better treatment response. The sequencing-based copy number analysis revealed locus-specific gains or losses including those previously reported in prostate cancers, such as 8q gains, AR amplifications, PTEN losses and TMPRSS2-ERG fusions. We found that overall PGA score was significantly higher in CRPC patients than in HSPC patients (p<0.0005), possibly reflecting increased tumor burden during the disease progression. Patients with low TEff index often demonstrated poor response to treatments, and patients with high initial PGA score or low TEff index tended to have poor overall survival. As examples of the PGA-based TEff associations with outcome we presented three CRPC patients including two with neuro-endocrine origin (small cell carcinoma). Comparison of mutational profiles between pre- and post-treatments showed that treatments caused more diverse gene mutations, in particular, in the genes involving androgen biosynthesis, AR activation, DNA repair and chemotherapy resistance, suggesting subclonal evolution of heterogeneous tumor genomes in response to these treatments. Our results strongly support the feasibility of using non-invasive liquid biopsy as a potential powerful tool to study biological mechanisms underlying therapy-specific resistance and monitor disease progression in the advanced prostate cancer. Further studies are needed to develop and validate the genomics-based PGA score and TEff index for clinical applications. Citation Format: Liang Wang, Shu Xia, Meijun Du, Rachel Ditmar, Tiezheng Yuan, Yongchen Guo, Yuan Wang, Adam Lee, Michael Tschannen, Elizabeth Worthey, Howard Jacobs, Chiang-Ching Huang, Manish Kohli. Plasma genetic and genomic abnormalities predict treatment response and clinical outcome in advanced prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5231. doi:10.1158/1538-7445.AM2015-5231

Abstract Background: Blood-based biopsies can be used as a non-invasive method to recover both Circulating Tumor Cells (CTCs) and Circulating Cancer Associated Macrophage-Like cells (CAMLs) from the blood of cancer patients. CAMLs are a newly-defined circulating immune cell type, described as a subtype of circulating stromal cells, known to engulf tumor cells/debris. CAMLs are identified by their large size, 30-300 microns, and aberrant nuclear structure. We studied the peripheral blood of cancer patients to ascertain the prevalence, specificity and sensitivity of CAMLs in relation to their disease status at presentation, including a variety of benign and malignant diseases. We supply evidence that this previously unidentified circulating cell can be used as a screening tool to detect solid tumors in numerous malignancy subtypes in all disease stages. Methods: CellSieveTM microfilters were used to isolate CAMLs from 7.5 mL of whole peripheral blood. The pore size of CellSieveTM is 7 microns, capable of isolating both CTCs and CAMLs based on size. Collected cells were fixed, permeabilized, and stained with DAPI and antibodies against cytokeratin 8, 18 and 19, CD14, and CD45. CAMLs were defined as enlarged, multinuclear cells with diffuse cytoplasmic cytokeratin staining, and/or CD14+, and/or CD45+. CTCs were defined as filamentous cytokeratin cells that are CD45-. Study 1: Blinded peripheral blood samples from 61 cancer patients were tested, including Stage I-IV breast, pancreatic, lung and prostate cancers, and newly diagnosed, untreated patients. Non-blinded blood samples from 30 healthy volunteers served as controls. Study 2: Double blinded peripheral blood samples from 20 patients with abnormal mammogram results were drawn at time of tissue biopsy and screened. After analysis, unblinding showed a distribution of malignant breast disease (n = 10), benign (n = 8), or atypical hyperplasia (AH) (n = 2). Results: Study 1: CAMLs were found in 94% patients with Stage III/IV cancers, 87% with Stage I/II cancers, and 89% of all patients, regardless of cancer type. Specifically, CAMLs were found in 77% of prostate, 100% of pancreatic, 80% of lung, and 100% of breast patient samples. CAMLs were not found in healthy control samples (sensitivity: 84-96%, Specificity: 100%, PPV: 100%, NPV: 88-97%). Study 2: CAMLs were found in 8 patients with malignant disease, 2 with benign disease, and 2 with AH (sensitivity: 80%, Specificity: 75%, PPV: 80%, NPV: 75%) Conclusions: We present evidence that CAMLs can be used as a non-invasive blood based biopsy, to detect the anatomical presence of solid malignancies and potentially pre-malignant lesions. The presence of CAMLs in newly diagnosed patient samples suggests their use as a blood biomarker for early stage cancer screening in a broad population. Preliminary data from a double blind breast cancer study suggests that CAMLs may differentiate between benign and malignant breast diseases. Citation Format: Daniel Adams, Katherine Alpaugh, Massimo Cristofanilli, Stuart Martin, Saranya Chumsri, Raymond C. Bergen, Susan Tsai, Martin Edelman, Peixuan Zhu, Shuhong Li, Olga V. Makarova, Platte T. Amstutz, Cha-Mei Tang, Jeffrey R. Marks. Cancer associated macrophage-like cells as a blood-based biomarker for the screening of solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5165. doi:10.1158/1538-7445.AM2015-5165

Abstract Glioblastoma (GBM), a highly invasive primary brain tumour, remains an incurable disease (1). Angiogenesis, the formation of new blood vessels, is a defining feature of GBM (2). Targeting GBM angiogenesis with Bevacizumab (Bev) is associated with improved progression free survival, but may also significantly enhance local tumour invasion (3). RhoGTPases and their activators, guanine nucleotide exchange factors (GEFs), play central roles in the invasive process (4). Herein, we sought to identify and target GEFs of importance in meditating GBM invasion with a view to improving Bev response. We report a novel strategy by which GBM tumours invade and proliferate via overexpression of the GEF beta-PIX which was shown to be increased at the invasive edge in 74% of GBM tumours assessed (n = 19) compared with tumour core (5). We have further shown that siRNA-mediated knockdown of beta-PIX in GBM patient-derived xenograft cell cultures and cell lines resulted in decreased cell invasion in 3D, cell proliferation and survival assays in vitro. Furthermore, we have uncovered a role for beta-PIX expression in endothelial cell function, as knockdown of beta-PIX inhibits HUVEC cell migration in vitro. To interrogate in vivo effects, we established an imagable orthotopic model of invasive GBM in mice, by stereotactically implanting GBM cells stably overexpressing firefly luciferase (U87R-GFP-luc2). beta-PIX-(human and mouse) specific siRNA or scrambled control siRNA (2 × 10 μg i.c.) were delivered to tumours using an alphaVbeta3 integrin targeting-(RGD peptide conjugated)-nanoparticle (InVivoPlex Aparna Bio Corp, Rockville, MD). Animals were treated with- or without- Bev (6 × 10mg/kg i.p. over 12 days). Initial data has shown that animals treated with beta-PIX siRNA nanoparticles in combination with Bev had an improved median survival of 100 days compared with Bev- alone treated mice (median: 88 days). Our data may support the future clinical use of a beta-PIX-targeting nanoparticle therapeutic, to improve Bev outcomes in GBM. 1. Kohler BA et al J Natl Cancer Inst. 2011; 103, 714-736 2. Jain RK et al Nat Rev Neurosci. 2007; 8(8):610-22 3. Norden AD et al Neurology 2008; 70: 779-787 4. Murray D et al Brit J Can 2014; 110(5):1307-15 5. Hoelzinger DB et al Neoplasia 2005; 7(1): 7-16 Citation Format: David W. Murray, Philip O'Halloran, Monika Jarzabek, Brian MacCarthy, Jann N. Sarkaria, Raymond M. Schiffelers, Marc Symons, Annette T. Byrne. Pre-clinical assessment of a novel anti-invasion nanoparticle therapeutic in combination with bevacizumab for the treatment of glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5093. doi:10.1158/1538-7445.AM2015-5093

Abstract Glioblastoma (GBM) is a highly lethal CNS malignancy in adults with a median survival of about 15 months. Effective therapeutic control of GBM is thwarted by the invasive nature of the tumor, which prevents complete surgical removal and results in tumor recurrence. Thus, there is a dire need to develop innovative approaches to target the invasive tumor cells for improved treatment of this disease. Expression of TROY, a member of the TNFR family, increases with glial tumor grade and inversely correlates with patient survival. Increased expression of TROY stimulates glioblastoma cell invasion and increases resistance to temozolomide (TMZ) and radiation treatment. We have demonstrated that TROY forms a novel complex with EGFR and modulates EGFR survival signaling. IHC analysis of GBM specimens showed that tumors with elevated TROY expression had a statistically positive correlation with increased EGFR expression. TROY expression enhanced EGFR phosphorylation, stabilized EGFR surface expression in the presence of ligand, and significantly increased EGF-stimulated GBM cell invasion. Consistent with invasion, co-expression of TROY with EGFR increased cell survival to TMZ. Moreover, we have shown that in cells treated with EGF, the TROY and ErbB4 receptor are recruited to the EGFR complex. Although the role of ErbB4 in GBM is not well defined, inhibition of ErbB4 expression suppressed tumor growth and invasion in multiple tumor subtypes, decreased EGFR surface retention, and limited EGF-induced cell migration. These data suggest that the TROY-EGFR-ErbB4 complex may represent an unappreciated therapeutic target to inhibit glioma invasion and decrease therapeutic resistance. Citation Format: Alison Roos, Zachary Mayo, Jean Kloss, Serdar Tuncali, Harshil Dhruv, Michael E. Berens, Joseph C. Loftus, Nhan L. Tran. TROY-EGFR signaling complex mediates glioblastoma cells invasion and survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 506. doi:10.1158/1538-7445.AM2015-506

Abstract KRAB-Associated Protein 1 (KAP1) is a master regulator for genome stability, heterochromatin formation, and target gene silencing. Many studies have suggested that post-translational modification is required for KAP1 to execute pleiotropic effects. Here, we have identified the physical interaction between KAP1 and SIRT1, which is a widely recognized NAD+-dependent deacetylase in DNA damage response. Therefore, the objective of this study was to characterize the functional significance of KAP1 deacetylation in DNA damage repair signaling. In this study, SIRT1 was identified as a major interacting partner of KAP1 via biochemical approaches. Inhibition of SIRT1 activity by shRNA or selective inhibitor was able to upregulate the total acetylation level on KAP1, suggesting that KAP1 is a novel target of SIRT1. The major SIRT1-regulated KAP1 deacetylation sites (Lys266, Lys377, Lys469, and Lys770) were detected by quantitative mass spectrometric analysis. Next, to explore the functional significance of KAP1 deacetylation in NHEJ-mediated repair, KAP1 depleted cells reconstituted with wild-type KAP1 or 4KR acetylation mutant were used in the in vivo end-joining reporter system. Compare to wild-type KAP1, the 4KR mutant presented higher activity in promoting NHEJ efficiency. Furthermore, 4KR mutant showed stronger binding affinity to 53BP1, a crucial NHEJ promoting factor, and stabilized the 53BP1 foci formation in response to DNA damage. In summary, this study unambiguously established KAP1 as a target of SIRT1 and that the deacetylation of KAP1 on Lys266, Lys377, Lys469, and Lys770 stabilizes the formation of 53BP1 foci on sites of double-strand breaks, thereby promotes NHEJ-mediated repair pathway. Citation Format: Yi-Hui Lin, Zhenkun Lou. SIRT1 regulates the function of the KRAB-associated protein 1 (KAP1) in DNA damage response signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4979. doi:10.1158/1538-7445.AM2015-4979

Abstract Background Genomic intratumor heterogeneity is an increasingly recognized phenomenon in various solid tumors and is now extensively being investigated by next-generation sequencing (NGS) approaches. However, most methods are not designed to resolve the complexity of mixed cell populations. Therefore important information may be lost, eventually leading to assumptions that may not reflect the true nature of the disease. We have therefore developed the method of Multiparameter Ploidy Profiling (MPP). We applied this technology on tumor specimens from malignant melanoma and from non-small cell lung cancer (NSCLC). Methods MPP involves isolation of nuclei from tissues, multiparameter flow-sorting of tumor populations by DNA content and specific tumor markers, and profiling genomes using high resolution array comparative genomic hybridization (aCGH) and NGS. As some tumors are diploid by flow cytometry, specific lineage markers, e.g. SOX10 (melanoma) and TTF1 (NSCLC) were used to separate these from normal cells. Fluorescence-in-situ hybridization (FISH) and immunohistochemistry (IHC) were performed on sorted nuclei and on whole tissue sections to validate results. Results MPP allowed us to sort different tumor populations within a single tumor specimen, while excluding non-neoplastic contamination in downstream analysis with aCGH and NGS. We separated three different tumor populations from a single primary NSCLC with a TTF1 multiparameter sort. We detected significant differences in copy numbers, e.g. a CDKN2A homozygous deletion only present in the diploid tumor population as well as differences in allelic frequencies of gene mutations (e.g. EGFR exon 20). Further sequencing of the diploid TTF1 negative population proved to be concordant to genomic DNA obtained from a tumor free lymph node. Finally, we used the population-specific genomic data in order to infer the clonal relationship of all tumor populations and to postulate the evolution of these tumor populations within single tumor specimens. Conclusions The power of MPP consists of sorting pure tumor populations and of deciphering the level of intratumor heterogeneity based on ploidy. Most importantly MPP enables the separation of diploid tumor populations from diploid, benign stromal cells that can serve as reference for genomic profiling studies, where germline DNA is otherwise not available. To our knowledge, diploid tumor populations were for first time purifyed from normal cells in a multiparameter sort and genomically characterized. We also demonstrated that the analysis of diploid tumor populations within a single biopsy is crucial for the understanding of the clonal composition and evolution of a given tumor. MPP on multiple biopsies from individual patients is necessary to deeply investigate the clonal evolution and the behavior of a diploid tumor population and their potential impact on metastasis and therapy response. Citation Format: Thomas Lorber, Sabrina Rau, Valeria Perrina, Michael Barrett, Christian Ruiz, Lukas Bubendorf. Multiparameter ploidy profiling: a powerful tool to investigate the genomics of diploid tumor populations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4924. doi:10.1158/1538-7445.AM2015-4924