Citations

Abstract INTRODUCTION Next generation sequencing (NGS) of patients has significantly changed our ways to study cancer genomics as it provides precise estimates of gene expression, fusion transcripts, expressed single nucleotide variants (eSNVs), splice variants and copy number variants. The Breast Cancer Genome Guided Therapy (BEAUTY) is an ongoing clinical study in which RNA sequencing (RNAseq) and whole exome sequencing (WES) are performed prior to, during and after neoadjuvant chemotherapy. Here we report the use of these sequencing technologies to investigate gene expression levels and mutational profiles in a triple negative breast cancer (TNBC) patient enrolled in BEAUTY whose disease did not respond to neoadjuvant paclitaxel and anthracycline/cycphosphamide. METHODS Computational approaches were used to integrate WES and RNAseq data obtained before therapy (V1T), after 12 weekly paclitaxel treatments (V2T) and after anthracycline-based regimen at surgical resection (V3T) to study a single patient with persistent TNBC disease after neoadjuvant chemotherapy. RESULTS Using RNA-Seq data, we identified an inter-chromosomal fusion transcript between chromosome 20 and 22 (GNAS-TTC38) that was highly expressed at V1T, V2T and V3T. We also identified intra-chromosomal fusion transcripts that were expressed at two time points, such as fusion transcript (KANSL1-ARL17A) on chromosome 17 for V1T and V2T and fusion transcript (RBM12B-LINC00535) on chromosome 8 for V2T and V3T time points. Several gene expression changes were also observed. Gene expression analysis of V1T, V2T and V3T tumors was performed. Differential temporal gene expression profiles of 9884 genes that were significant and varying at different time points were obtained for pathway analysis. Pathway analysis of 9884 genes identified up regulation and down regulation of several transcription factors with a fold change of 2x or more. When compared to blood, DNA tumor and RNA-Seq data, we identified 81 common somatic eSNVs that were expressed in both V1T and V2T time points and we are in the process of investigating V3T data. We found alterations of key transporter domains (CD225, Coatamer_beta_C, DUF2435, Dynamitin, EI24, GLTP, LMF1, Porin_3, V-ATPase_C) in our V1T and V2T SNV data. Similar to gene expression analysis, we are in the process of obtaining the list of mutations at various time points to identify driver and passenger mutation candidate genes for this specific TNBC patient. CONCLUSIONS Our initial time-series analysis of eSNV, fusion transcripts and gene expression data demonstrate that intensive analysis for individual patients is feasible. Further investigation of drug transporters and transcription regulators may help develop personalized treatment strategies for patients with disease resistant to current regimens. Citation Format: Krishna R. Kalari, Xiaojia Tang, Kevin J. Thompson, Douglas W. Mahoney, Poulami Barman, Jason P. Sinnwell, Hugues Sicotte, Peter Vedell, Steven N. Hart, Travis J. Dockter, Katie N. Jones, Amy L. Conners, Ann M. Moyer, Daniel W. Visscher, Jia Yu, Bowen Gao, Sarah A. McLaughlin, John A. Copland, Alvaro Moreno-Aspitia, Donald W. Northfelt, Richard J. Gray, Vera J. Suman, Jeanette E. Eckel Passow, Jean-Pierre A. Kocher, Eric D. Wieben, Gianrico Farrugia, Cloann G. Schultz, James N. Ingle, Richard Weinshilboum, Matthew P. Goetz, Liewei Wang, Judy C. Boughey. Analysis of sequencing data to identify potential drug targets for an individual newly diagnosed with basal breast cancer who failed to respond to current standard neoadjuvant chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4185. doi:10.1158/1538-7445.AM2014-4185

Abstract Background Cancer genomic investigations have identified recurrent genomic aberrations critical for cancer initiation, progression, and metastases. However, these investigations are typically performed in isolation, and the effects of treatment on the clonal selection of tumor cells are mostly unknown. We hypothesized that molecular profiling of residual tumors after neoadjuvant chemotherapy (NAC) would identify new drug targets/pathways in patients at high risk for disease recurrence. To better identify clonal populations of resistant breast cancer cells, we utilized DNA content-based flow sorting of nuclei to identify and isolate clonal populations for aCGH and next generation sequencing (NGS) both before and after NAC. Methods The Breast Cancer Genome Guided Therapy Study (BEAUTY) (NCT 02022202) is a prospective study of patients with high-risk breast cancer treated with neoadjuvant 12 weekly paclitaxel (T) +/- trastuzumab followed by 4 cycles of anthracycline based chemotherapy. Tumor tissue from baseline, residual disease from surgery, distant metastases, and patient derived xenografts (PDX) are obtained for cell sorting by DNA ploidy, aCGH, RNA and exome sequencing. Results: 140 patients have been enrolled, 104 have completed surgery and 30 unique PDX have been established corresponding to 26 patients prior to chemotherapy and 4 from residual disease at surgery. Baseline exome and RNA sequencing is complete in 140. Currently, genomic analyses of flow sorted matched baseline, surgical, PDX, and distant disease samples are available in 6 patients. Substantial genomic variation was observed in the surgical sample compared to the primary tumor including gain of oncogenic drivers (EGFR) and loss of negative regulators (ATG5) (Table). The PDX recapitulated these events with excellent fidelity compared to the corresponding human tumor. In patients with TNBC, RNA seq obained from matched samples demonstrated changes in immune related pathways. Evaluation of drug targets/pathways identified in the resistant tumors are ongoing using the PDX and sequencing of the remaining matched baseline/surgical disease will be reported. Clonal changes occurring over time in patients with residual disease or disease recurrence after NACTumor SubtypeResidual Cancer BurdenDisease StatusClonal Aberration Changes (baseline and post NAC)TNBC (AR subtype)3Contralateral lymph node recurrence at 150 days2p25.2 - p25.1 amplicon lost at recurrence; 6p21.32 -p21.31 amplicon lost at recurrenceTNBC (BL 1)3Bone/liver/lymph node recurrence at 135 days5q11.2 amplicon gained at surgery; 12p13.33 - p13.2 amplicon gained at surgeryTNBC (BL 1)3Disease-free at day 1506q21 amplicon lost at sugeryTNBC (BL 2)0Brain recurrence at 390 daysChr 2 chromothripsis at surgeryLuminal B3Progression during chemotherapy7p12.1 - p11.2 amplicon gained at surgeryLuminal HER23Disease-free at day 3609q33.2 amplicon lost at surgery; 15q13.1 - q13.3 amplicon lost at surgery; 15q22.2 - q24.1 amplicon lost at surgery Conclusions: We observed substantial evolutionary changes in residual breast tumors remaining after NAC. Our findings suggest that a comprehensive assessment of the mutational landscape that has evolved during NAC can inform drug development in high risk breast cancer patients. Citation Format: Matthew P Goetz, Michael T Barrett, Krishna R Kalari, Vera J Suman, Sarah A McLaughlin, Alvaro Moreno-Aspitia, Ann M Moyer, Donald W Northfelt, Richard J Gray, Jason Sinnwell, Douglas Mahoney, Poulami Barman, Peter Vedell, Xiaojia Tang, Kevin Thompson, Travis Dockter, Katie Jones, Sara J Felten, Amy Conners, Jeanette Eckel-Passow, Hughes Sicotte, Steven N Hart, Jia Yu, Daniel W Visscher, Eric D Wieben, Cloann Schultz, Minetta C Liu, James N Ingle, Liewei Wang, Richard W Weinshilboum, Judy C Boughey. Impact of neoadjuvant chemotherapy on the clonal composition of breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD3-3.

Abstract Background. Follicular lymphoma (FL) is an indolent B-cell malignancy with a variable clinical course that may transform to more aggressive forms of lymphoma. Previous modestly powered GWAS have reported multiple FL susceptibility loci in the HLA class I and II regions at 6p21.32-33, but novel non-HLA susceptibility loci and the genetic architecture of FL associations in the HLA region remain to be elucidated. Methods. We conducted the largest GWAS of FL to date consisting of 2,142 cases and 6,221 controls of European ancestry from 22 studies scanned on the Illumina OmniExpress. After imputation of common SNPs using IMPUTE2 and 1,000 Genomes Project v3 data, we conducted a meta-analysis of these data with two previous FL GWAS (n=586 cases, 1,537 controls) and replicated top hits in an additional 629 cases and 4,283 controls. Expression quantitative trait loci (eQTL) analyses were performed to evaluate the effects of associated variants on gene expression. Classical HLA allele imputations and stepwise regression analyses are underway to further characterize the HLA associations. Results. In the joint meta-analysis, the strongest association with FL risk was observed in the HLA region, where a large number of SNPs showed genome-wide significance (P<5x10-8 to P= 1.84x10-84), particularly in HLA class II at 6p21.32 where several HLA eQTLs were identified (P < 0.05). Outside HLA, we identified four novel loci associated with FL at 11q23.3 (CXCR5, rs4938573; P=3.14x10-15), 11q24.3 (ETS1, rs4937362; P=3.33x10-9), 3q13.33 (CD86, rs2681416; P=9.79x10-11) and 3q28 (LPP, rs6444305, P=2.55x10-8). These novel susceptibility loci were located in four biologically relevant genes involved in immune regulation: CXCR5 and its ligand, CXCL13, are involved in guiding B-cells into the B-cell zones of secondary lymphoid organs as well as T-cell migration. ETS1 is expressed in lymphoid cells and regulates immune cell function including differentiation, survival, and proliferation. CD86 and CD80 are classic members of the B7 costimulatory pathway that is important in maintaining immune function, suppression of autoimmunity and antitumor surveillance. LPP encodes a LIM domain-containing protein of the zyxin family and participates in cell adhesion, cell migration, proliferation and transcription dynamics. Conclusions. This large GWAS provides further support for the important role of common genetic variation in non-HLA genes and further evidence of the key role that HLA immune-regulatory genes play in the pathogenesis of FL. Citation Format: Christine F. Skibola, Sonja I. Berndt, James R. Cerhan, Zhaoming Wang, Joseph Vijai, Lucia Conde, Paul de Bakker, Sophia S. Wang, Claire M. Vajdic, Brenda M. Birmann, Susan L. Slager, James McKay, Paige M. Bracci, Alexandra Nieters, Qing Lan, Angela R. Brooks-Wilson, Martha S. Linet, Demetrius Albanes, John J. Spinelli, Roel C.H. Vermeulen, Mark P. Purdue, Meredith Yaeger, Lauren R. Teras, Silvia de Sanjose, Alain Monnereau, Simon Crouch, Jia Nee Foo, Henrik Hjalgrim, Gianluca Severi, Brian K. Link, Kimberly A. Bertrand, Yawei Zhang, Karin E. Smedby, Stephen J. Chanock, Nathaniel Rothman, NHL GWAS Consortium. Meta-analysis of genome-wide association studies identifies novel susceptibility loci for follicular lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5072. doi:10.1158/1538-7445.AM2014-5072

Abstract INTRODUCTION Patient derived xenografts (PDX) may better reflect individual patient (pt) tumor biology; however, the feasibility of collecting PDX from percutaneous tumor biopsies (PTB) in the neoadjuvant setting is unknown. Furthermore, drug response phenotypes observed in PDX have not been prospectively compared to the corresponding pt clinical outcomes. METHODS The Breast Cancer Genome Guided Therapy Study (BEAUTY) is a prospective Mayo study of pts with high-risk breast cancer treated with neoadjuvant weekly paclitaxel (T) +/- trastuzumab followed by anthracycline based chemotherapy. PTB (at baseline) and residual surgical tissue (after all chemotherapy) are obtained for next generation sequencing (NGS) and PDX. Tumor biopsies (1-2 cores from 14 gauge needle) were implanted with Matrigel <1 hour of collection in the flanks of NOD-SCID or NSG mice. Low dose estradiol was supplemented in the drinking water. Primary outgrowth rate was defined as PDX tumor volume >50 mm3. Take rate was defined as development of at least 1 stably transplantable xenograft line/pt. To determine whether clinical T response assessed by MRI corresponded with in vivo T response, pretreatment PDX from 5 pts were injected into NOD-SCID mice (20 mice per pt PDX) and when tumors reached 100-200mm3, mice were randomized to no treatment vs T (20 mg/kg, ip. every 3-4 days). Two of these 5 patients had a MRI response defined as >30% decrease in longest lesion. RESULTS Pretreatment PTB from 81 unique pts were implanted in 251 mice (2-4 mice/pt). PDX outgrowth rates were 33.3% (27/81 pts) and 22 stable PDX were established (overall take rate 27.2%). Take rates were as follows: triple negative breast cancer (46%; 13/28); HER2 (27%; 6/22), Luminal B (13%; 3/22), and luminal A (0%; 0/9). Residual surgical tumor (after all treatment) from 17 pts was injected into 85 mice (average 5 mice/pt) and the initial outgrowth rate was 23% (4/17) with 3 stably transplantable lines established. PDX, derived from pretreatment PTB of 5 pts (2 responders and 3 non-responders), were assessed for in vivo T response. The size of the T treated group was significantly smaller than the no treatment group for the PDX derived from the 2 clinical responders, with complete disappearance of tumor by 18 days. In contrast, the PDX derived from the 3 clinical non-responders had no evidence for T response. CONCLUSIONS We have demonstrated the feasibility of using PTB to establish PDX in a prospective neoadjuvant clinical study and have demonstrated similar T drug response phenotypes in in the PDX as seen in the corresponding pt. These data suggest that PDX generated prospectively may be useful for biomarker validation and the development and individualization of new drug therapy. Funded by the Mayo Clinic Center for Individualized Medicine and the Mayo Clinic Cancer Center Citation Format: Jia Yu, Ping Yin, Bowen Gao, Jason P. Sinnwell, Ann M. Moyer, Daniel W. Visscher, Amy L. Conners, Travis J. Dockter, Krishna R. Kalari, Xiaojia Tang, Kevin J. Thompson, Hugues Sicotte, Douglas W. Mahoney, Steven N. Hart, Peter T. Vedell, Poulami Barman, Katie N. Jones, Sarah A. McLaughlin, John A. Copland, Alvaro Moreno Aspitia, Donald W. Northfelt, Richard J. Gray, Vera J. Suman, Jeanette E. Eckel Passow, Eric D. Wieben, James N. Ingle, Zhenkun Lou, Gianrico Farrugia, Richard Weinshilboum, Matthew P. Goetz, Judy C. Boughey, Liewei Wang. Feasibility of using percutaneous tumor biopsies from a prospective neoadjuvant breast cancer study to develop patient derived xenografts and assess in vivo chemotherapy sensitivity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1195. doi:10.1158/1538-7445.AM2014-1195

Abstract Background: Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related death in the United States; however, it remains poorly characterized in surgical populations. To better inform perioperative transfusion practice, and to help mitigate perioperative TRALI, the authors aimed to better define its epidemiology before and after TRALI mitigation strategies were introduced. Methods: This retrospective cohort study examined outcomes of adult patients undergoing noncardiac surgery with general anesthesia who received intraoperative transfusions during 2004 (n = 1,817) and 2011 (n = 1,562). The demographics and clinical characteristics of transfusion recipients, blood transfusion descriptors, and combined TRALI/possible TRALI incidence rates were evaluated. Univariate analyses were used to compare associations between patient characteristics, transfusion details, and TRALI mitigation strategies with TRALI/possible TRALI incidence rates in a before-and-after study design. Results: The incidence of TRALI/possible TRALI was 1.3% (23 of 1,613) in 2004 versus 1.4% (22 of 1,562) in 2011 (P = 0.72), with comparable overall rates in males versus females (1.4% [23 of 1,613] vs. 1.2% [22 of 1,766]) (P = 0.65). Overall, thoracic (3.0% [4 of 133]), vascular (2.7% [10 of 375]), and transplant surgeries (2.2% [4 of 178]) carried the highest rates of TRALI/possible TRALI. Obstetric and gynecologic surgical patients had no TRALI episodes. TRALI/possible TRALI incidence increased with larger volumes of blood product transfused (P < 0.001). Conclusions: Perioperative TRALI/possible TRALI is more common than previously reported and its risk increases with greater volumes of blood component therapies. No significant reduction in the combined incidence of TRALI/possible TRALI occurred between 2004 and 2011, despite the introduction of TRALI mitigation strategies. Future efforts to identify specific risk factors for TRALI/possible TRALI in surgical populations may reduce the burden of this life-threatening complication.

Abstract Background: Transfusion-associated circulatory overload (TACO) is a leading cause of transfusion-related fatalities, but its incidence and associated patient and transfusion characteristics are poorly understood. To inform surgical transfusion practice and to begin mitigating perioperative TACO, the authors aimed to define its epidemiology. Methods: In this retrospective cohort study, the medical records of adult patients undergoing noncardiac surgery with general anesthesia during 2004 or 2011 and receiving intraoperative transfusions were screened using an electronic algorithm for identification of TACO. Those patients who were screened as high probability for TACO underwent rigorous manual review. Univariate and multivariate analyses evaluated associations between patient and transfusion characteristics with TACO rates in a before-and-after study design. Results: A total of 2,162 and 1,908 patients met study criteria for 2004 and 2011, respectively. The incidence of TACO was 5.5% (119 of 2,162) in 2004 versus 3.0% (57 of 1,908) in 2011 ( P < 0.001), with comparable rates for men (4.8% [98 of 2,023]) and women (3.8% [78 of 2,047]) ( P = 0.09). Overall, vascular (12.1% [60 of 497]), transplant (8.8% [17 of 193]), and thoracic surgeries (7.2% [10 of 138]) carried the highest TACO rates. Obstetric and gynecologic patients had the lowest rate (1.4% [4 of 295]). The incidence of TACO increased with volume transfused, advancing age, and total intraoperative fluid balance (all P < 0.001). Conclusions: The incidence of perioperative TACO is similar to previous estimates in nonsurgical populations. There was a reduction in TACO rate between 2004 and 2011, with incidence patterns remaining comparable in subgroup analyses. Future efforts exploring risk factors for TACO may guide preventive or therapeutic interventions, helping to further mitigate this transfusion complication.

Abstract Pancreatic cancer is the 10th most common cancer and 4th most common cause of cancer mortality in the United States. A genome wide association study has revealed pancreatic cancer susceptibility loci on chromosomes 13q22.1, 1q32.1 and 5p15.33. The chr13q22.1 region contains the most significant of all risk SNPs, rs9543325, (P= 3.27 x 10−11) which is located in a gene desert. The nearest genes are KLF5, KLF12, PIBF1, DIS3 and BORA, which range in distance from 265kb to 586kb, respectively, from the most significant variants. Given the high linkage disequilibrium across this risk locus, there are many candidate functional variants to consider. Imputation in the region did not improve the signal but gave a set of highly correlated SNPs which likely includes the functional variant(s). In an effort to identify the functional variant(s) we performed eQTL analyses to test the association between the genotypes of these candidate SNPs and expression of nearby genes. Among 100 normal pancreatic tissue samples, DIS3 showed the strongest association with SNPs in our risk locus (P-values as low as 0.0004). Mutations in DIS3 have been identified in acute myeloid leukemia and multiple myeloma, and its expression has been correlated with metastatic potential in colorectal cancer, suggesting this gene could be important in pancreatic cancer biology. Chromosome Conformation Capture (3C) was performed to test for physical interactions between the risk locus and nearby genes. This assay confirmed the three dimensional proximity of the risk locus and DIS3 promoter. Sub-regions of the risk locus were then cloned upstream of a minimal promoter controlling luciferase expression to assay for potential allele specific enhancer/silencer activity. This assay revealed a sub-region downstream of rs9543325 that causes allele-specific silencing. These results suggest that a sub-region of the chr13q22.1 risk locus has allele-specific effects on DIS3 expression, which may have implications in the susceptibility to pancreatic cancer. Citation Format: Jason Hoskins, Abdisamad Ibrahim, Jinping Jia, Irene Collins, Hemang Parikh, Gloria M. Petersen, Laufey Amundadottir. Functional analysis of the chr13q22.1 pancreatic cancer risk locus suggests allele-specific effects on DIS3 expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3137. doi:10.1158/1538-7445.AM2013-3137

AbstractBackgroundRegulatory qualification of a biomarker for a defined context of use provides scientifically robust assurances to sponsors and regulators that accelerate appropriate adoption of biomarkers into drug development.MethodsThe Coalition Against Major Diseases submitted a dossier to the Scientific Advice Working Party of the European Medicines Agency requesting a qualification opinion on the use of hippocampal volume as a biomarker for enriching clinical trials in subjects with mild cognitive impairment, incorporating a scientific rationale, a literature review and a de novo analysis of Alzheimer's Disease Neuroimaging Initiative data.ResultsThe literature review and de novo analysis were consistent with the proposed context of use, and the Committee for Medicinal Products for Human Use released an opinion in November 2011.ConclusionsWe summarize the scientific rationale and the data that supported the first qualification of an imaging biomarker by the European Medicines Agency.