Citations

Abstract Background Individual carrying germline mutations within BRCA1 or BRCA2 gene confers well-established increased risk of developing breast and ovarian cancers in the life-time than the general population. Recent advancement in clinical oncology and pharmacology discovered that poly ADP ribose polymerase (PARP) inhibitors are particularly effective in treating cancer that is homologous recombination deficient (HRD) through synthetic lethality, irrespective of the primary tumor site or the type of the BRCA1/2 inactivation variant. Methods A dedicated next-generation sequencing (NGS) panel was designed to interrogate the mutational status of BRCA1/2 and 18 other HRD-associated genes. Using a cohort of 522 clinical samples and artificially engineered cell line samples, we evaluated the mutation detection proficiency for single nucleotide variant (SNV) and small insertion or deletion (Indel) variants. BRCA1/2 inactivation is often the consequence of large genomic rearrangement (LGR) within the gene and phenotypically shown as exon-level copy number variant (CNV). Given the technical challenge of accurate exon-level CNV detection within a small panel, we developed a noise-reduction model based on a pool of normal (PON) samples for CNV result analysis. We further designed droplet digital polymerase chain reaction (ddPCR) for each exon of BRCA1/2 to verify the exonic CNV findings. Results Using a comparator assay as the gold standard, the BRCA1/2 panel achieved an accuracy of 96.7%, a precision of 98.9%, and a lower limit of detection of 3% variant allelic frequency (VAF) for SNV and Indel. The CNV pipeline achieved 97.8% sensitivity and 99.7% specificity for exonic CNV events that were supported by at least one piece of secondary evidence, out-performing publicly available software. The ddPCR verification further enhances assay accuracy and provides a significantly improved statistically confidence for the final CNV calls unparalleled by any other experimental methodologies. Conclusions Using a large cohort of clinical samples, the authors demonstrated the proficiency of a targeted NGS approach for variant and LGR detection of BRCA1/2 and the other genes of the DNA damage repair pathway. An effective method was developed to suppress the noise within the NGS-derived CNV profile, which is exaggerated by the small panel size and short targeted regions. The authors would also like to call the researcher's attention to a critical caveat within the CNV testing results generated from NGS-based methods. Citation Format: Xiangyuan Ma, Hua Bao, Zhili Chang, Yong Wu, Changwan Du, Rong Gao, Yu Song, Ryan S. Robetyore, Xue Wu, Yang W. Shao. Technical validation of a NGS-based analysis pipeline for BRCA1/2 variants and large genomic rearrangements detection [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4268.

Abstract In vivo positron emission tomography (PET) imaging is a key modality to evaluate disease status of brain tumors. In recent years, tremendous efforts have been made in developing PET imaging methods for pediatric brain tumors. Carbon-11 labelled tryptophan derivatives are feasible as PET imaging probes in brain tumor patients with activation of the kynurenine pathway, but the short half-life of carbon-11 limits its application. Using a transgenic mouse model for the sonic hedgehog (Shh) subgroup of medulloblastoma, here we evaluated the potential of the newly developed 1-(2-[18F]fluoroethyl)-L-tryptophan (1-L-[18F]FETrp) as a PET imaging probe for this common malignant pediatric brain tumor. 1-L-[18F]FETrp was synthesized on a PETCHEM automatic synthesizer with good chemical and radiochemical purities and enantiomeric excess values. Imaging was performed in tumor-bearing Smo/Smo medulloblastoma mice using a Perkin-Elmer G4 PET-X-Ray scanner. Medulloblastoma tumors showed significant and specific accumulation of 1-L-[18F]FETrp. 1-L-[18F]FETrp also showed significantly higher tumor uptake than its D-enantiomer 1-D-[18F]FETrp. The uptake of 1-L-[18F]FETrp in normal brain tissue was low, suggesting that 1-L-[18F]FETrp may prove a valuable PET imaging probe for the Shh subgroup of medulloblastoma and possibly other pediatric and adult brain tumors. Citation Format: Xuyi Yue, Yangchun Xin, Hua Li, Zhiqin Li, Hancheng Cai, Sigrid A. Langhans. Tumor-specific PET imaging with an 18F-labeled tryptophan analogue in a transgenic medulloblastoma mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1652.

AbstractThis report by the Radiation Oncology Discipline of Children's Oncology Group (COG) describes the practice patterns of pediatric image‐guided radiotherapy (IGRT) based on a member survey and provides practice recommendations accordingly. The survey comprised of 11 vignettes asking clinicians about their recommended treatment modalities, IGRT preferences, and frequency of in‐room verification. Technical questions asked physicists about imaging protocols, dose reduction, setup correction, and adaptive therapy. In this report, the COG Radiation Oncology Discipline provides an IGRT modality/frequency decision tree and the expert guidelines for the practice of ionizing image guidance in pediatric radiotherapy patients.

Abstract Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous entity of B-cell lymphoma. Cell-of-origin (COO) classification of DLBCL is required in routine practice by the World Health Organization classification for biological and therapeutic insights. Genetic subtypes uncovered recently are based on distinct genetic alterations in DLBCL, which are different from the COO subtypes defined by gene expression signatures of normal B cells retained in DLBCL. We hypothesize that classifiers incorporating both genome-wide gene-expression and pathogenetic variables can improve the therapeutic significance of DLBCL classification. To develop such refined classifiers, we performed targeted RNA sequencing (RNA-Seq) with a commercially available next-generation sequencing (NGS) platform in a large cohort of 418 DLBCLs. Genetic and transcriptional data obtained by RNA-Seq in a single run were explored by state-of-the-art artificial intelligence (AI) to develop a NGS-COO classifier for COO assignment and NGS survival models for clinical outcome prediction. The NGS-COO model built through applying AI in the training set was robust, showing high concordance with COO classification by either Affymetrix GeneChip microarray or the NanoString Lymph2Cx assay in 2 validation sets. Although the NGS-COO model was not trained for clinical outcome, the activated B-cell–like compared with the germinal-center B-cell–like subtype had significantly poorer survival. The NGS survival models stratified 30% high-risk patients in the validation set with poor survival as in the training set. These results demonstrate that targeted RNA-Seq coupled with AI deep learning techniques provides reproducible, efficient, and affordable assays for clinical application. The clinical grade assays and NGS models integrating both genetic and transcriptional factors developed in this study may eventually support precision medicine in DLBCL.

AbstractLarge observational data networks that leverage routine clinical practice data in electronic health records (EHRs) are critical resources for research on coronavirus disease 2019 (COVID-19). Data normalization is a key challenge for the secondary use of EHRs for COVID-19 research across institutions. In this study, we addressed the challenge of automating the normalization of COVID-19 diagnostic tests, which are critical data elements, but for which controlled terminology terms were published after clinical implementation. We developed a simple but effective rule-based tool called COVID-19 TestNorm to automatically normalize local COVID-19 testing names to standard LOINC (Logical Observation Identifiers Names and Codes) codes. COVID-19 TestNorm was developed and evaluated using 568 test names collected from 8 healthcare systems. Our results show that it could achieve an accuracy of 97.4% on an independent test set. COVID-19 TestNorm is available as an open-source package for developers and as an online Web application for end users (https://clamp.uth.edu/covid/loinc.php). We believe that it will be a useful tool to support secondary use of EHRs for research on COVID-19.

PURPOSE Electronic health records (EHRs) are created primarily for nonresearch purposes; thus, the amounts of data are enormous, and the data are crude, heterogeneous, incomplete, and largely unstructured, presenting challenges to effective analyses for timely, reliable results. Particularly, research dealing with clinical notes relevant to patient care and outcome is seldom conducted, due to the complexity of data extraction and accurate annotation in the past. RECIST is a set of widely accepted research criteria to evaluate tumor response in patients undergoing antineoplastic therapy. The aim for this study was to identify textual sources for RECIST information in EHRs and to develop a corpus of pharmacotherapy and response entities for development of natural language processing tools. METHODS We focused on pharmacotherapies and patient responses, using 55,120 medical notes (n = 72 types) in Mayo Clinic’s EHRs from 622 randomly selected patients who signed authorization for research. Using the Multidocument Annotation Environment tool, we applied and evaluated predefined keywords, and time interval and note-type filters for identifying RECIST information and established a gold standard data set for patient outcome research. RESULTS Key words reduced clinical notes to 37,406, and using four note types within 12 months postdiagnosis further reduced the number of notes to 5,005 that were manually annotated, which covered 97.9% of all cases (n = 609 of 622). The resulting data set of 609 cases (n = 503 for training and n = 106 for validation purpose), contains 736 fully annotated, deidentified clinical notes, with pharmacotherapies and four response end points: complete response, partial response, stable disease, and progressive disease. This resource is readily expandable to specific drugs, regimens, and most solid tumors. CONCLUSION We have established a gold standard data set to accommodate development of biomedical informatics tools in accelerating research into antineoplastic therapeutic response.

Abstract GX15-070 is a small-molecule antagonist of the BH3-binding groove of the Bcl-2 family of proteins and can thus induce apoptosis by inhibiting the interaction between pro- and anti-apoptotic proteins. In addition, GX15-070 activates a cellular pathway, which leads to the activation of the pro-apoptotic BH3-only protein Bim, a natural cellular inhibitor of anti-apoptotic members. Together, the direct inhibition of anti-apoptotic Bcl-2 family proteins and the activation of pro-apoptotic Bim by GX15-070 can sensitize tumor cells to apoptosis. Bcl-2 family members, particularly Bcl-XL and Mcl-1 have been implicated in the survival of myeloma cells and may be responsible, in part, for drug resistance. Consequently, GX15-070 was tested against the MMRC 12 validation cell line panel, a panel of 12 standardized and annotated human myeloma cell lines (HMCLs). Western blots preformed on 8 HMCLs confirmed expression of Bcl-2, Mcl-1 and/or Bcl-XL. Basal levels of Bcl-2 and MCl-1 were relatively similar across all HMCLs studied whereas levels of Bcl-XL were variable GX15-070 inhibited the viability of all 12 myeloma cell lines as determined by MTT assay. The mean IC50 was 215 nM; range (6.4-678nM) and sensitivity did not correlate with level of Bcl-2, MCL-1 or Bcl-XL expression. On the other hand cytotoxicity to normal human blood lymphocytes, stroma and bone marrow CFUs was not observed at concentrations which were effectively cytotoxic to HMCL. Time course experiments demonstrate that apoptosis and cleavage of caspase 3 began by 12 hours and continued to increase over a 96 hour period. Co-culture with human bone marrow stroma cells (BMSCs) failed to protect HMCL from GX15-070 induced cytotoxicity. Similarly, potent MM growth factors, Interleukin 6 (IL-6) and Insulin like growth factor (IGF)-1 did not confer resistance to GX15-070. Of interest, overexpression of Bcl-2 has been shown to attenuate PS-341-induced cell death (Mitisades); however, the combination of PS-341 and GX15-070 did not reveal synergistic interaction in preliminary results. Studies looking at other drug combinations are planned. Finally, GX15-070 produced cytotoxic responses in the first primary myeloma sample tested with further testing ongoing. Mouse xenograft studies are also ongoing and will be presented. These studies indicate extensive pre-clinical activity of GX15-070 in multiple myeloma and set the stage for planned Phase II clinical trials.

Abstract The importance of aberrant signal transduction in the development and progression of cancers including multiple myeloma (MM) is well recognized, although the detailed regulation of signaling networks in relation to oncogenic mutations remains incompletely understood. However, this subject is becoming increasing relevant to clinical oncology due the rapid development of biological-based therapies that can target signaling pathways. Analytical methods that can be applied to clinical samples to measure complex populations of cells and phenotype them for multiple activation states are now feasible due to the recent advances in intracellular signal techniques, flow cytometry and phospho-antibodies. Using multi-parameter flow cytometry, we monitored phospho-protein responses to activators/cytokines and signal transduction inhibitors in a panel of 14 extensively characterized human MM cell lines with heterogeneous molecular abnormalities. A panel of 4-phospho-specific antibodies: anti-pS6, anti-ERK1/2, anti-pAKT and anti-pSTAT3 that represent downstream target genes of signaling pathways known to play central roles in myelomagenesis were used in the initial analysis. Sixteen conditions (basal, inhibitor, activator alone, activator and inhibitor) were studied to generated 64 readouts designed to survey altered signal transduction in the 14 cell lines. We collected data on unstimulated cells and cells stimulated for 7–10 min with IL-6, IGF-1 or FGF or cells inhibited with the signal transduction inhibitors U0126 (MEK), rapamycin (mTOR) or LY294002 (PI3-K). Repeat measurements were collected and the technique and monoclonal antibodies displayed a high level of reproducibly. In contrast, the basal, cytokine and inhibitor responses between cell lines varied considerably reflecting the molecular heterogeneity at the level of signaling responses. The protocols that have been refined and validated in myeloma cell lines are now being applied to primary bone marrow samples from MM patients. Although the same size thus far is small, phospho-protein responses among primary CD138 positive myeloma cells show considerable induction and variance of AKT, MAPK, STAT3 and pS6 phosphorylation. In some MM tumor samples treatment with inhibitors suggest constitutive activation of these signaling pathways while in others the nodes remain activable with phosphorylation above the basal state following stimulation. The data further suggests that MAPK phosphorylation following aFGF stimulation displays significant variance among MM samples and correlates with the detection of t(4;14) translocation by FISH analysis. Additional samples are being assessed and correlation of phospho-protein responses with clinical parameters and cytogenetics will be reported. The data demonstrate that multi-parameter flow cytometry can be applied to myeloma tumor samples to study the phospho-proteome and that considerable heterogeneity exists at the level of signaling responses.

8025 Background: The Src/Stat 3 pathway has been implicated in the pathogenesis of several malignancies including melanoma. In the current study we evaluated the prevalence of Src and Stat3 activation in human melanoma and the effect of Src inhibitors alone or in combination with chemotherapeutic agents on melanoma cell lines. Methods: Activation of Src was measured by staining for Src-phosphotyrosine 416 (P-Src) and Stat3 by phosphotyrosine 705 Stat3 (P-Stat3). Immunohistochemistry was performed with anti-pStat3-Y705 and anti-pSrc-Y416 antibodies (Cell Signaling, Beverly, MA) on 3 melanoma subtypes. Staining of tumor cells for P-Stat3 was scored by percentage of stained cells as follows: 0 (no staining), 1 (1–25%), 2 (26–50%), 3 (>50%), and intensity 0–3 (0: absent, 1: low, 2: moderate, and 3: high). The sum of these was the composite P-Stat3 score: 0 (0), 1 (1–2), 2 (3–4), and 3 (5–6). P-Src staining was scored by intensity. Melanoma cell lines, A-375, SK-Mel-5 and SK-Mel-28 were exposed to Src inhibitors, SKI-606, PD 180970 and BMS 354825 alone or in combination with standard chemotherapy agents and IC-50 was determined by MTT assay at 24 and 72 hours. Results: 35 biopsies were stained. Median age was 66 years (range 23–90); 60% were women. 48% of all melanoma stained express P-Src; all samples express pStat3. We found a positive correlation between P-Src and P-Stat3 (Spearman coefficient=0.346, p=0.042). 46% stained only for pStat3 and 6% did not stain for either P-Src or P-Stat3. Most positively stained biopsies stained weakly for P-Src and moderately or strongly for pStat3. Src inhibitors inhibited the proliferation of melanoma cell lines; furthermore Src inhibitor treatment synergized with platinum and topoisomerase I inhibitors but not temozolomide. Conclusions: 1) Immunohistochemistry shows variable expression of P-Src and P-Stat3 in human melanoma 2) All tumors staining for P-Src also stain for P-Stat3 and the strength of staining is significantly related. 3) This data may be useful in planning future clinical trials with Src inhibitors in melanoma. [Table: see text] [Table: see text]