Search

CDK4/6 inhibitors such as palbociclib in combination with endocrine therapy (ET) have remarkablyimproved the outcome of patients with ER+/HER2- metastatic breast cancer (MBC). However, manypatients are intrinsically resistant to CDK4/6i therapy, and those who respond eventually acquireresistance. Although high baseline CCNE1 expression and rare alterations in RB1 and FAT1 geneshave been shown to be associated with CDK4/6i resistance, the molecular mechanisms of CDK4/6iresistance are complex and remain poorly understood. To better understand and overcome CDK4/6iresistance, we performed multi-omics profiling of paired tumor biopsies from ER+/HER2- MBCpatients treated with palbociclib combined with ET. Tumor biopsies taken at pre-treatment, on-treatment, and progressive disease (PD) from 71 patients were profiled using whole-exomesequencing (WES), whole-transcriptome sequencing (RNA-Seq) and IHC analysis. Ourcomprehensive analysis identified several tumor intrinsic molecular markers associated with worsePFS, including the Luminal B subtype (p=0.012, HR=2.593), BRCA1/2 pathogenic mutation (p=0.012,HR=2.67) and mutation signatures linked to APOBEC enzymatic activity (p=0.002, HR=3.19).Conversely, the estrogen response signature (p=0.006, HR=0.43) was associated with favorableprognosis. Unsupervised analysis revealed a cluster of tumors enriched in homologousrecombination deficiency (HRD) linked genomic scars that was associated with poor prognosis(p=0.005, HR=2.49). Of note, these HRD-high tumors responded even more poorly to treatment whenco-occurring with TP53 somatic mutations. Integrative analysis further identified three poorprognosis clusters (IC2-4) enriched in Luminal B, proliferative and HRD features when compared tothe favorable prognosis cluster (IC1).Comparing baseline vs. PD samples, we observed a pattern of post-treatment enrichment for the poorprognosis markers. In addition, breast cancer-associated genes such as BRCA1/2, TP53 and PTENharbored a higher prevalence of genomic alterations including somatic mutation, amplification,. deletion and gene fusion at PD. Cell cycle gene expression and signatures also markedly increased atPD compared to baseline whereas estrogen response signatures decreased. Upon diseaseprogression, tumors had frequently switched to molecular subtypes with aggressive and estrogenindependent characteristics, demonstrating high plasticity in response to CDK4/6i and ET treatment.These patterns of acquired resistance were validated by IHC analysis of cyclins E1 and E2, Ki67 andpRb. To investigate the genomic alterations responsible for acquired resistance, we compared 21paired baseline and PD samples. We observed that PD-specific RB1 loss-of-function events occurredwith higher prevalence than previously reported, underscoring a major role of cell cycle de-regulation in conferring resistance to CDK4/6 inhibition. In this prospective longitudinal multi-omicsstudy, we identified novel candidate biomarkers that can be used to improve prediction of responseto CDK4/6i. In addition, we derived new insights into the molecular mechanisms of drug resistanceto palbociclib plus ET that will help guide therapeutic strategies and drug development inHR+/HER2− MBC. Citation Format: Zhengyan Kan, Seock-Ah Im, Kyunghee Park, Ji Wen, Kyung-Hun Lee, Yoon-La Choi, Won-Chul Lee, Ahrum Min, Vinicius Bonato, Seri Park, Sripad Ram, Dae-Won Lee, Ji-Yeon Kim, Su Kyeong Lee, Won-Woo Lee, Jisook Lee, Miso Kim, Scott L. Weinrich, Han Suk Ryu, Tae Yong Kim, Stephen Dann, Diane Fernandez, Jiwon Koh, Song Yi Park, Shibing Deng, Eric Powell, Rupesh Kanchi Ravi, Jadwiga Bienkowska, Paul A. Rejto, Woong-Yang Park, Yeon Hee Park. Serial genomic profiling reveals molecular mechanisms of breast cancer resistance to palbociclib [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD2-08.

Background Intrinsic breast cancer molecular subtyping (IBCMS) provides significant prognostic information for patients (pts) with breast cancer (BC) treated with chemotherapy, human epidermal growth factor receptor 2 (HER2) targeted therapies, and endocrine therapies (ETs). Classifying tumors into intrinsic subtypes to determine optimal treatment is often applied using PAM50, commercially known as Prosigna. Meanwhile, Absolute Assignment of Breast Cancer Intrinsic Molecular Subtypes (AIMS) computational method was trained to predict PAM50-based IBCMS. As the PAM50 algorithm was developed to capture the major subtypes in a general pt population, clinicopathologic distribution of the study cohort and technology platform calibration should be considered in IBCMS analyses. This study compared different next-generation sequencing technologies and methodologic approaches of PAM50 on tumor samples from 2 randomized trials of postmenopausal women with estrogen receptor-positive (ER+)/HER2-negative (HER2-) BC. Methods PALOMA-2 is a double-blind, randomized study of first-line palbociclib (PAL) + letrozole (LET) for ER+/HER2- advanced BC (ABC). Tumor samples from consented pts were subtyped using the validated RUO PAM50 assay (ruoProsigna, NanoString); results were compared with published subtype results using AIMS on EdgeSeq Oncology Biomarker Panel (HTG Molecular Diagnostics). PALLET is a phase 2, randomized trial of PAL+LET as neoadjuvant therapy in pts with ER+ HER2- BC. Baseline frozen tumor biopsies underwent whole transcriptome mRNA-sequencing (mRNA-seq). IBCMS was performed using AIMS; PAM50 subtyping was performed on data normalised with subgroup-specific gene centering and microarray-RNA-sequencing calibration. Results In PALOMA-2, 222 pts had both ruoProsigna and AIMS data; an overall 54% agreement rate between methods was observed, with 46% (56/121) of Luminal B (LumB) subtype by ruoProsigna assigned as Luminal A (LumA) by AIMS and 67% (6/9) of basal-like by ruoProsigna as HER2-enriched (HER2-E) by AIMS (Table 1). In PALLET, 224 pts had mRNA-seq data; a 69% agreement between the two approaches (AIMS and PAM50) was observed, with only 4% (2/49) of LumB assigned as LumA by AIMS but 17% (26/156) and 16% (25/156) of LumA considered LumB or normal-like by AIMS, respectively. Progression-free survival (PFS) by ruoProsigna-derived subtype in PALOMA-2 showed that PAL+LET benefited all pts but those with a basal-like subtype (Table 2). With AIMS, PAL+LET provided a PFS benefit in pts with LumA and LumB subtypes, but was less effective in the HER2-E subtype. Conclusion Intrinsic subtyping has potential clinical utility. PAL+ET should be considered for ER+/HER2- ABC, except possibly in pts with a basal-like tumor, consistent with previous reports. A standardized clinical PAM50 assay and bioinformatics approach should be used as discrepancies in gene expression platforms and algorithms lead to different results and could misguide treatment decisions. Clinical trial identification: Pfizer (NCT01740427) Table 1.Intrinsic Subtyping by IBCMS MethodsPALOMA-2PALLETMethodruoProsignaPAM50 mRNAseqAIMS BasalHER2LumALumBGrand TotalBasalHER2LumALumBNormalGrand TotalBasal-like, n (%)1 (11)NANANA13 (75)0001 (8)4HER2-E, n (%)6 (67)6 (30)6 (8)13 (10)311 (25)3 (100)8 (5)6 (12)1 (8)19LumA, n (%)NA2 (10)60 (83)56 (46)1180097 (62)2 (4)099LumB, n (%)2 (22)12 (60)3 (4)52 (43)690026 (17)41 (84)067Normal-like, n (%)NANA3 (4)NA30025 (16)010 (83)35Grand Total9 (100)20 (100)72 (100)121 (100)2224 (100)3 (100)156 (100)49 (100)12 (100)224NA=Not available Table 2.Median PFS statistics by subtype in PALOMA-2PAL+LET PFS, monthsPBO+LET PFS, monthsHazard Ratio(95% CI)P ValueruoProsignaBasal-like8.2 (n=5)3.6 (n=4)0.39 (0.09-1.77)0.206HER2-E11.0 (n=12)5.1 (n=8)0.41 (0.15-1.11)0.071LumA37.2 (n=52)13.6 (n=20)0.42 (0.21-0.84)0.011LumB27.6 (n=79)13.8 (n=42)0.63 (0.40-1.00)0.049AIMSBasal-likeNANANANAHER2-E16.4 (n=21)8.4 (n=10)0.82 (0.32-2.1)0.684LumA30.6 (n=84)16.5 (n=34)0.56 (0.33-0.95)0.029LumB19.3 (n=41)8.8 (n=28)0.39 (0.23-0.67) Citation Format: Maggie Cheang, Mitch Dowsett, Mothaffar Rimawi, Stephen Johnston, Samuel Jacobs, Judith Bliss, Katherine Pogue-Geile, Lucy Kilburn, Zhou Zhu, Eugene F. Schuster, Hui Xiao, Lisa Swaim, Shibing Deng, Dongrui R. Lu, Eric Gauthier, Jennifer Tursi, Dennis J. Slamon, Hope S. Rugo, Richard S. Finn, Yuan Liu. Impact of using cross-platform gene expression profiling technologies and computational methods for intrinsic breast cancer subtyping in PALOMA-2 and PALLET [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD2-07.

Select laboratory abnormalities by maximum CTCAE grade

Gene signatures in baseline peripheral blood samples associated with prognosis in follicular lymphoma patients treated with rituximab and utomilumab. Higher, normalized enrichment scores (NES) correspond to poorer survival. NES were derived from gene set enrichment analysis with genes pre-ranked by hazard ratio from multivariate Cox proportional-hazards model stratified by dose levels and adjusting for age, sex, race, stage, and whether patients had progressed on prior rituximab-containing therapy. Gene sets assessed were derived from the Molecular Signatures Database (MSigDB) hallmark collection (Liberzon et al, Cell Syst 2015;1:417-25). Only significant cell populations are shown (FDR-adjusted q value

Pharmacokinetic parameters for utomilumab in combination with rituximab (Cycle 1)

(A) Levels of soluble 4-1BB/CD137 and (B) changes in CD8+ T cells from Cycle 1 day 1, in individual patients by dose group.

Background: We have previously identified two potentially predictive signatures of palbociclib resistance: the RBsig, composed of E2F1/E2F2 dependent genes, which is correlated with genetic loss of RB1, and the ratio between the gene expression levels of CCNE1 to RB1 (CCNE1/RB1). Both signatures have been previously tested in vitro and in neoadjuvant studies with palbociclib. The present analysis aims to explore the prognostic and predictive role of RBsig and CCNE1/RB1 in the pivotal phase III randomized trials PALOMA-2 and PALOMA-3. Materials and methods: Gene expression data from the PALOMA-2 and PALOMA-3 datasets were generated using the HTG EdgeSeq Oncology BM Panel, as previously described. Of the 87 genes composing RBsig, 46 were available within the EdgeSeq dataset and were used for the analyses; CCNE1 and RB1 were both available. RBsig was calculated as the mean of the Z-score scaled gene expression (log) of the 46 genes; CCNE1/RB1 was computed as the log ratio between the mRNA expression of CCNE1 and RB1. High and low values of RBsig and CCNE1/RB1 were defined based on the third quartile (Q3) as cutoff or as continuous variables. The prognostic/predictive effect of the signatures in terms of PFS was tested using Cox proportional hazard models and the Wald test. Results: The 46-genes RBsig versus the original signature showed excellent correlation in the METABRIC dataset (R=0.99), confirming its reliability as a surrogate of the original RBsig using EdgeSeq data. In both PALOMA-2 and PALOMA-3, RBsig high was significantly associated with a worse outcome compared to RBsig low in the palbociclib arm but not in the control arm [PALOMA-2: HR 1.4 (95% CI 1.0, 2.0) p=0.029 for palbociclib arm; HR 1.1 (95% CI 0.7, 1.6) p=0.71 for control arm. PALOMA-3: HR 1.7 (95% CI 1.1, 2.6) p=0.01 for palbociclib arm; HR 1.2 (95% CI 0.7, 1.9) p= 0.49 for control arm]. However, in both studies RBsig was not predictive of palbociclib resistance both when considered as a continuous variable and when dichotomized at Q3. Similarly to RBsig, in PALOMA- 3 patients with CCNE1/RB1 high tumors treated in the palbociclib arm showed a significantly worse outcome compared to those with CCNE1/RB1 low but this effect was not observed in those treated in the control arm [HR 1.6 (95% CI 1.1- 2.5) p= 0.03 for palbociclib arm; HR 1.2 (95% CI 0.7, 1.9) p=0.5 for control arm]. In addition, CCNE1/RB1 as a continuous variable was predictive of palbociclib benefit in PALOMA-3 (interaction p= 0.047). These effects were not observed in PALOMA-2. Conclusions: RBsig is a prognostic biomarker in patients treated with palbociclib, suggesting it may help in patients’ risk stratification. CCNE1/RB1 is predictive of palbociclib benefit in PALOMA-3, but not in PALOMA-2 probably due to the different patient populations and characteristics. Further studies of these biomarkers in patients treated with CDK4/6 inhibitors in the metastatic as well in the adjuvant setting are warranted. Citation Format: Luca Malorni, Matteo Benelli, Yuan Liu, Shibing Deng, Zhe Zhang, Cristina Guarducci, Angela Leo, Agostina Nardone, Francesca Galardi, Emanuela Risi, Erica Moretti, Dario Romagnoli, Chiara Biagioni, Marta Paoli, Laura Biganzoli, Ilenia Migliaccio. Prognostic and predictive role of RBsig and CCNE1/RB1 gene-expression signatures in patients with advanced breast cancer treated with palbociclib in combination with endocrine therapy in the PALOMA-2 and 3 trials [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-17.

Background: POLARIS is a prospective, real-world study of palbociclib in patients with hormone receptor–positive (HR+)/human epidermal growth factor receptor 2–negative (HER2–) advanced breast cancer (ABC) in the United States and Canada. We present results from analyses of serial circulating tumor gene alteration profiles from patients treated with palbociclib in the first and second or later lines of ABC treatment to illustrate the potential mutagenic drivers of resistance (intrinsic/acquired) and carrier-mutations (intrinsic/maintained). Methods: The clinical database cutoff date was March 30, 2022. Patients in the biomarker analysis group provided consent for serial blood sample collection, received ≥1 dose of initial palbociclib combination treatment, and had ≥1 circulating tumor DNA (ctDNA) measurement available. The Guardant360 platform with somatic single-nucleotide variants in complete or critical exons of 73 genes was used. Tumor gene alteration profiles (at baseline, on-treatment at Cycle 2 Day 1 [C2D1], and at end of treatment [EOT]) were evaluated. Cox proportional hazard models were used to estimate hazard ratios and 95% CIs. Results: Patient samples (n=345) were analyzed and gene alterations were detected in 85% of baseline samples (n=337), 72% of on-treatment samples (n=280), and 85% of EOT samples (n=104). Most frequently altered genes were PIK3CA (38%), TP53 (28%), and ESR1 (15%) at baseline; TP53 (28%), PIK3CA (24%), and NF1 (10%) at C2D1; and TP53 (40%), PIK3CA (40%), and ESR1 (33%) at EOT. Most frequent gene amplifications (amp) were detected in CCND1 (8.3%), FGFR1 (7.7%), and EGFR (5.9%) at baseline; FGFR1 (5.0%), CCND1 (4.3%), and EGFR (3.2%) at C2D1; and CCND1 (13.5%), FGFR1 (9.6%), and EGFR (9.6%) at EOT. Baseline mutations of ESR1 and PIK3CA led to shorter real-world progression-free survival (rwPFS) than wild-type (hazard ratio [95% CI], 1.99 [1.38, 2.86] and 1.67 [1.24, 2.25], respectively). Baseline amp of CCND1 and FGFR1 also led to shorter rwPFS than wild-type (2.13 [1.36, 3.34] and 1.93 [1.20, 3.10]). Acquired mutations in ESR1, ATM, and RB1 were observed at EOT. Most frequently acquired ESR1 mutations at EOT were D538G, Y537N, and Y537S. Patients with all mutations cleared at C2D1, had longer rwPFS than those without (hazard ratio [95% CI], 0.58 [0.41, 0.83]). Conclusion: Patients with mutated ESR1 and PIK3CA or CCND1 and FGFR1 amp at baseline had shorter rwPFS than patients with wild-type genes. Genotyping analysis of progression ctDNA highlights the emergence of mutations in estrogen receptor and cell cycle pathways under selective therapeutic pressure and could guide monitoring and therapeutic sequencing for patients with HR+/HER2– MBC. ClinicalTrials.gov: NCT03280303 Citation Format: Debu Tripathy, Joanne L. Blum, Shibing Deng, Steven L. McCune, Kamal Patel, Yao Wang, Shailendra Lakhanpal, Meghan S. Karuturi, Zhe Zhang, Chetan Deshpande, Monica Z. Montelongo, Eric Gauthier, Yuan Liu, Gabrielle B. Rocque, Aditya Bardia. Circulating Tumor DNA Genotyping of Intrinsic and Acquired Gene Alterations in Patients With Advanced Breast Cancer Receiving the Cyclin-Dependent Kinase 4/6 Inhibitor Palbociclib: Biomarker Results from POLARIS [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-22.

Background: The PENELOPE-B (NCT01864746) and PALLAS (NCT02513394) trials are large prospective, randomized, phase III trials that evaluated adjuvant palbociclib (PAL) + endocrine treatment (ET) vs ET in patients with hormone receptor–positive/human epidermal growth factor receptor 2–negative (HER2–) early breast cancer (EBC). Both studies did not meet the primary endpoint of improving invasive disease-free survival (iDFS). We conducted biomarker analyses to identify patients who might benefit from PAL + ET in EBC. Methods: Resected tumor tissue was collected from consenting patients. Gene expression analyses were conducted using the HTG EdgeSeq Oncology Biomarker Panel including 2549 genes. Based on 91 genes from the HTG panel, the intrinsic molecular subtypes were calculated using Absolute Intrinsic Molecular Subtyping (AIMS). Potential predictive treatment biomarkers were established in PENELOPE-B (n=906 with resected tissue) as the development set using an outcome-oriented approach based on iDFS with a selection procedure that maximized the log-rank statistic to estimate a standard Z score–based optimal cutoff. Independent validation was conducted on PALLAS (n=2085; PENELOPE-B-like with resected tissue and HTG data). Hazard ratios and corresponding 95% CIs were calculated using the Cox proportional hazards model, and iDFS distributions between treatment arms were compared using the log-rank test. Interaction between treatment and biomarker status was assessed. Results: Patient baseline characteristics were well balanced, with no differences in iDFS between the intent-to-treat set and the biomarker set for both trials. Approximately 73% of patients (PENELOPE-B [n=663] and PALLAS [n=1516]) had luminal A subtypes whereas only 7.1 % (PENELOPE-B [n=64]) and 8.3 % (PALLAS [n=172]) had a luminal B subtype. AIMS subtypes showed overall similar prognostic patterns for iDFS between PENELOPE-B and PALLAS. The biomarker-defined subgroup found in PENELOPE-B with optimal cutoff demonstrated a preferential benefit from PAL + ET (n=364 [96 events]; hazard ratio [95% CI], 0.63 [0.42, 0.95]; P=0.025). Independent validation of the PALLAS subgroup using the pre-defined optimal cutoff confirmed a significant benefit from PAL + ET (n=916 [70 events]; 0.55 [0.34–0.90]; P=0.015) while not in the rest of the patients (interaction p=0.0025). Significant treatment effects remained (0.55 [0.34–0.89]; P=0.015) after adjusting for the randomization stratification factors of PALLAS. Conclusions: The composite predictive biomarker defined from PENELOPE-B was independently validated in a prospectively defined retrospective analysis of a subset of patients selected from PALLAS. The composite biomarker identified a subset of EBC patients deriving benefit from the addition of PAL to ET. This patient stratification approach can potentially be applied to future adjuvant clinical trials for treatment of hormone receptor–positive/HER2– EBC. Citation Format: Sibylle Loibl, Carsten Denkert, Yuan Liu, Erik S. Knudsen, Angela DeMichele, Zhe Zhang, Julia Teply-Szymanski, Martin Filipits, Peter A. Fasching, Michael Gnant, Shibing Deng, Marija Balic, Federico Rojo, Mark Watson, Chetan Deshpande, Nicholas Turner, Otto Metzger, Kathy Puyana Theall, Agnieszka Witkiewicz, Olga Valota, W. Fraser Symmans, Erica L. Mayer. Development and Validation of a Composite Biomarker Predictive of Palbociclib + Endocrine Treatment Benefit in Early Breast Cancer: PENELOPE-B and PALLAS Trials [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD17-05.

Background: In the randomized, two-part, phase 3 COLUMBUS study (NCT01909453), encorafenib (enco) + binimetinib (bini) and enco alone improved 5-year progression-free survival (PFS) and overall survival (OS) vs vemurafenib (vem) in patients (pts) with advanced BRAF V600E/K-mutant melanoma. ctDNA offers a less invasive alternative to tissue biopsy for identifying mutations in pts with advanced melanoma and may have prognostic value. We evaluated whether ctDNA from pts enrolled in COLUMBUS Part 1 correlate with pts’ efficacy outcomes. Methods: In COLUMBUS Part 1, 577 pts were randomized 1:1:1 to enco + bini, enco, or vem. 650 plasma samples were collected at baseline (Cycle 0 or Cycle 1 Day 1; all arms) and during treatment (Cycle 2 Day 1 [C2D1]; enco + bini and enco arms); 426 samples from COLUMBUS Part 1 were successfully analyzed using the GuardantOMNI assay. Analyses were exploratory with no multiplicity adjustments. Results: Survival outcomes in the biomarker cohort were similar to those in the intent-to-treat population. ctDNA was detected in 279 (96%) pts at baseline and 107 (79%) pts at C2D1. 92% concordance was observed between two plasma genomics assays (Inostics and Guardant) and 75% between tumor and plasma with respect to BRAF mutation status. BRAF V600 variant allele frequency (VAF) at baseline was prognostic for PFS and OS (P Conclusions: These exploratory analyses of Part 1 of the COLUMBUS study suggested that treatment with enco + bini significantly reduced BRAF V600 VAF, which was prognostic for PFS and OS at both baseline and C2D1. ctDNA provides a powerful tool for understanding the molecular basis for response and resistance to treatment and may impact future treatment decisions. Citation Format: Reinhard Dummer, Nuzhat Pathan, Zhou Zhu, Caroline Robert, Ana Arance, Jan Willem B. de Groot, Claus Garbe, Helen J. Gogas, Ralf Gutzmer, Ivana Krajsová, Gabriella Liszkay, Carmen Loquai, Mario Mandala, Dirk Schadendorf, Naoya Yamazaki, Michelle Edwards, Jean Cantey-Kiser, Alessandra di Pietro, Shibing Deng, Paolo A. Ascierto, Keith Flaherty. Molecular correlates of clinical benefit from circulating tumor DNA (ctDNA): Analysis of the COLUMBUS study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6403.

Purpose: In this phase I study (NCT01307267), we evaluated safety, pharmacokinetics, clinical activity, and pharmacodynamics of treatment with utomilumab plus rituximab in patients with relapsed/refractory follicular lymphoma (FL) and other CD20+ non-Hodgkin lymphomas (NHL). Patients and Methods: Primary objectives were to assess treatment safety and tolerability for estimating the MTD, using a modified time-to-event continual reassessment method, and selecting the recommended phase II dose (RP2D). Results: Sixty-seven patients received utomilumab (0.03–10.0 mg/kg every 4 weeks) and rituximab (375 mg/m2 weekly) in the dose-escalation groups or utomilumab (1.2 mg/kg every 4 weeks) plus rituximab in the dose-expansion cohort. No patient experienced dose-limiting toxicity. The MTD for utomilumab in combination with rituximab was not reached and estimated to be ≥10 mg/kg every 4 weeks. The majority of the utomilumab treatment-related adverse events (AE) were grade 1 to 2; the most common AE was fatigue (16.4%). The pharmacokinetics of utomilumab in combination with rituximab was linear in the 0.03 to 10 mg/kg dose range. A low incidence (1.5%) of treatment-induced antidrug antibodies against utomilumab was observed. The objective response rate was 21.2% (95% CI, 12.1%–33.0%) in all patients with NHL, including four complete and 10 partial responses. Analysis of paired biopsies from a relapsed/refractory FL patient with complete response showed increased T-cell infiltration and cytotoxic activity in tumors. Biomarker correlations with outcomes suggested that clinical benefit may be contingent on patient immune function. Conclusions: Utomilumab in combination with rituximab demonstrated clinical activity and a favorable safety profile in patients with CD20+ NHLs.

Increased gene copy number has been associated with a greater antiproliferative response upon genome editing, regardless of the true essentiality profile of the targeted gene. Many methods have been developed to adjust for genomic copy number technical artifacts. Existing methods use a two-step correction by pre-processing the data prior to the final analysis. It has been shown that two-step corrections can produce unreliable results, due to the creation of a correlation structure in the corrected data. If this structure is unaccounted for, gene-essentiality levels can be inflated or underestimated, affecting the False Discovery Rate (FDR). We propose a one-step correction using restricted cubic splines (RCS) to be a simpler alternative which reduces the bias in downstream analyses. Moreover, most existing methods combine guide-level results to yield gene-level estimates which can misrepresent the true gene essentiality profile depending on the guide-averaging method. Our model-based approach (RESCUE-MM) for copy number correction provides a more flexible framework that allows for guide-level essentiality estimation while accommodating more complex designs with grouped data. We provide comparisons to existing copy number correction methods and suggest how to include copy number adjustment in a one-step correction fashion in multiple experimental designs.

The dysfunction and apoptosis of vascular endothelial cells are the initiating links in the formation of atherosclerosis. N6-methyladenosine (m6A) is an extremely extensive RNA methylation modification and its abnormality leads to the occurrence of various human diseases. In this study, we explored the effects of demethylase α-ketoglutarate-dependent dioxygenase ALKB homolog 5 (ALKBH5) on TNF-α-induced apoptosis of human umbilical vein endothelial cells (HUVECs). In TNF-α-treated HUVECs, the expression of ALKBH5 was significantly decreased. ALKBH5 overexpression promoted the proliferation and inhibited the apoptosis in TNF-α-treated HUVECs, suggesting that ALKBH5 had a protective effect on cell damage induced by TNF-α. Importantly, ALKBH5 promoted the expression of Bcl-2 in HUVECs. Bcl2 overexpression reduced the expression of Gadd45, Bax, and p21, which are transcriptionally activated by p53. But the expression of p53 has not been significantly affected, indicating that Bcl2 might regulate the apoptosis by inhibiting p53 downstream targets. In addition, ALKBH5 overexpression significantly increased the level of pri-miR-7 and decreased the level of miR-7. In conclusion, ALKBH5 attenuated the TNF-α-induced cell injury via promoting Bcl2 expression. Our research expands the understanding of the progression mechanism of atherosclerosis and provides a potential strategy for the protection of vascular endothelial injury.

Early studies on the etiology and pathogenesis of hypertension have shown that it has a considerable association with inflammation and the immune response as well as periodontitis. Clinical studies have also shown that hypertension can promote the periodontal tissue destruction caused by periodontitis. However, the underlying mechanisms remain unclear. This study aimed to explore the possible mechanisms of how hypertension aggravates periodontitis. Treatment with or without the signal transducer and activator of transcription 1 (STAT1) inhibitor fludarabine was performed in an endothelial nitric oxide synthase gene knockout-related ( Nos3-/-) mouse model with the hypertension phenotype of periodontitis induced by bacteria. Micro–computed tomography, immunohistochemistry, Western blot, quantitative reverse transcription polymerase chain reaction, immunofluorescence, and ELISA were performed. We demonstrated that Nos3-/--related hypertension increases bone resorption and periodontal destruction in periodontitis lesion areas, which can be inhibited by the STAT1 inhibitor. Experimental data also showed that Nos3-/-significantly increased macrophage infiltration and proinflammatory cytokine expression in the periodontitis lesion area, which is dependent on the angiotensin II–induced STAT1 pathway. Inhibition of STAT1 in vivo can decrease the expression of proinflammatory cytokines and macrophage infiltration. Furthermore, data in this study showed that Nos3-/--related hypertension further downregulated the STAT3 anti-inflammatory function and its downstream chemokine expression in a STAT1-dependent manner. By applying RAW 264.7 and L929 cell lines and monocytes isolated from Nos3-/-mice, we confirmed that activation of the STAT1 pathway inhibits STAT3 and its downstream pathway and promotes inflammatory cytokine expression in vitro. Collectively, our current study demonstrated that STAT1 plays an indispensable role in the Nos3-/--related hypertension with aggravation of periodontitis, suggesting that STAT1 may be a key target for the treatment of periodontitis with hypertension.

Understanding how standard-of-care drug treatments affect tumor intrinsic biology and microenvironment is critical for elucidating drug resistance mechanisms and developing better combination therapies as well as new therapies. To characterize the effects of neoadjuvant chemotherapy (NAC) on the genome, transcriptome and tumor infiltrating leukocytes (TILs), we have conducted whole exome and whole transcriptome sequencing of a large longitudinal breast cancer cohort consisting of 146 cases and 281 paired tumor samples. In total, 52 (38%) patients achieved pathologic complete response (pCR) while 85 patients (62%) had residual disease with standard chemotherapy regimen. Tumor biopsies were collected for each patient at three time points – pre-treatment, three weeks after the first cycle of anthracycline and cyclophosphamide (AC) and at the time of surgery after 3 more cycles of AC followed by 4 cycles of taxane or taxane plus Herceptin in case of HER2+ subtype. We detected 5,955 protein-altering somatic mutations affecting 4,414 genes in pretreatment samples and 502 acquired mutations in surgery samples affecting 477 genes including 19recurrently mutated genes such as TP53 and NOTCH1. Across all subtypes, 4,346 genes were differentially expressed (DE) following NAC treatment and significantly enriched in pathways such as cell cycle, ER signaling, PI3K/mTOR, immune and metabolism. Expression-based virtual microdissection analysis indicated that NAC treatment induced an increase in the fractions of stromal and adjacent normal tissue compartment, consistent with observed reduction in tumor cellularity. To assess the NAC induced changes in the molecular landscape of these tumors, we compared molecular features including gene expression signatures, mutation prevalence and copy number alteration between three time points while adjusting for confounding effects of molecular subtype and tumor cellularity. We found that NAC induced dynamic changes in gene expression signatures associated with proliferation and immunomodulatory treatment response. We further validated the observed pattern of change in TILs through histopathology and digital imaging analyses. In pretreatment tumors, 116 genes were DE between patients with pCR vs. those with residual disease with significant enrichment in immune/inflammatory pathways. Further, pre-treatment TIL levels were found to be significantly associated with pCR, echoing previous reports in breast cancers that implicated anti-tumor immunity in mediating the efficacy of chemotherapies. Our analyses also revealed associations between NAC response and baseline genomic attributes such as genomic alterations that affect DNA damage repair pathways. Taken together, these results suggest that NAC induced a multitude of changes on the genomic landscape and immune microenvironment of breast cancers, some of which point to combination strategies with immunomodulatory therapies and therapies that target DNA damage repair. Citation Format: Kan Z, Lal S, Ding Y, Lee JE, Lee S-H, Lee SK, Yu JH, Choi Y-l, Kim SW, Nam SJ, Kim J-Y, Ram S, Powell E, Ching K, Cho SY, Bonato V, Deng S, Park W-Y, Rejto P, Bienkowska J, Park Y-H. Neoadjuvant chemotherapy alters the genomic landscape and immune microenvironment of breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD5-08.

The CDK4/6 inhibitor palbociclib reduces tumor growth by decreasing retinoblastoma (RB) protein phosphorylation and inducing cell cycle arrest at the G1/S phase transition. Palbociclib in combination with anti-hormonal therapy brings significant benefit to breast cancer patients. In this study, novel combination approaches and underlying molecular/cellular mechanisms for palbociclib were explored in squamous cell lung cancer (SqCLC), the second most common subtype of non-small cell lung cancer. While approximate 20% lung patients benefit from immunotherapy, most SqCLC patients who receive platinum-doublet chemotherapy as first-line treatment, which often includes a taxane, are still in need of more effective combination therapies. Our results demonstrated enhanced cytotoxicity and anti-tumor effect with palbociclib plus taxanes at clinically achievable doses in multiple SqCLC models with diverse cancer genetic backgrounds. Comprehensive gene expression analysis revealed a sustained disruption of pRB-E2F signaling by combination that was accompanied with enhanced regulation of pleiotropic biological effects. These included several novel mechanisms such as abrogation of G2/M and mitotic spindle assembly checkpoints, as well as impaired induction of hypoxia-inducible factor 1 alpha (HIF-1α). The decrease in HIF-1α modulated a couple key angiogenic and anti-angiogenic factors, resulting in an enhanced anti-angiogenic effect. This preclinical work suggests a new therapeutic opportunity for palbociclib in lung and other cancers currently treated with taxane based chemotherapy as standard of care.

Immunohistochemistry (IHC) assays play a central role in evaluating biomarker expression in tissue sections for diagnostic and research applications. Manual scoring of IHC images, which is the current standard of practice, is known to have several shortcomings in terms of reproducibility and scalability to large scale studies. Here, by using a digital image analysis-based approach, we introduce a new metric called the pixelwise H-score (pix H-score) that quantifies biomarker expression from whole-slide scanned IHC images. The pix H-score is an unsupervised algorithm that only requires the specification of intensity thresholds for the biomarker and the nuclear-counterstain channels. We present the detailed implementation of the pix H-score in two different whole-slide image analysis software packages Visiopharm and HALO. We consider three biomarkers P-cadherin, PD-L1, and 5T4, and show how the pix H-score exhibits tight concordance to multiple orthogonal measurements of biomarker abundance such as the biomarker mRNA transcript and the pathologist H-score. We also compare the pix H-score to existing automated image analysis algorithms and demonstrate that the pix H-score provides either comparable or significantly better performance over these methodologies. We also present results of an empirical resampling approach to assess the performance of the pix H-score in estimating biomarker abundance from select regions within the tumor tissue relative to the whole tumor resection. We anticipate that the new metric will be broadly applicable to quantify biomarker expression from a wide variety of IHC images. Moreover, these results underscore the benefit of digital image analysis-based approaches which offer an objective, reproducible, and highly scalable strategy to quantitatively analyze IHC images.

Macroautophagy (autophagy) is an essential cellular catabolic process required for survival under conditions of starvation. The role of autophagy in cancer is complex, context-dependent and at times contradictory, as it has been shown to inhibit, promote or be dispensable for tumor progression. In this study, we evaluated the contribution of the immune system to the reliance of tumors on autophagy by depleting autophagy-related 7 (ATG7) in murine tumor cells and grafting into immunocompetent versus immunodeficient hosts. Although loss of ATG7 did not affect tumor growth in vitro or in immunodeficient mice, our studies revealed that cancer cell reliance on autophagy was influenced by anti-tumor immune responses, including those mediated by CD8+ T cells. Furthermore, we provide insights into possible mechanisms by which autophagy disruption can enhance anti-tumor immune responses and suggest that autophagy disruption may further benefit patients with immunoreactive tumors.

Circulating tumor DNA (ctDNA) refers to small fragments of tumor DNA in the total cell free DNA (cfDNA) from cancer patients. Detecting ctDNA in plasma has provided a promising, non-invasive liquid biopsy approach that could be utilized for cancer diagnosis and treatments. Depending on many factors, the quantity of ctDNA can be very low in the total cfDNA especially in early stage cancers, which posted a great challenge for ultra-sensitive detection with high specificity. Previously, we published TNER, an effective noise reducing algorithm based on tri-nucleotide context (TNC) data from healthy subject samples to polish background noise before calling mutations. It offers great specificity in mutation detection however it might over-polish true mutations when ctDNA concentration is very low. To improve its detection sensitivity in samples with low ctDNA concentration, we developed TNER2, a significant extension of TNER with a refined background polishing process by leveraging somatic mutation patterns from known cancer mutation data. Since there is a distinct difference between TNC pattern in cancer and healthy sample, TNER2 enables a more sensitive analysis on ctDNA data for early detection of cancer.

Patient-Derived Xenograft (PDX) provides important preclinical model for pharmacological testing of oncology drug candidates. Molecular profiling of PDX tumors contributes to many areas of drug discovery from target discovery to development of clinical biomarker hypotheses and clinical trial design. We established a work flow to perform genomic and histopathology analyses of large numbers of PDX tumor models being made available for Pfizer internal research. To date we have generated whole-genome sequencing (WGS), whole-exome sequencing (WES) and whole transcriptome sequencing (RNA-Seq) data on PDX models spanning six cancer types including colon, pancreatic and breast cancers. Bioinformatics pipelines were developed to quantify gene expression and detect genetic alterations including mutation, copy number variations and gene fusions. A controlled evaluation study demonstrated that in silico classification of NGS reads into human/mouse origins is more effective than laboratory-based methods for removing mouse tissue contamination. Comparative analyses of molecular profiles from PDX and primary tumors of the same cancer origins suggest that important patterns of gene expression are retained by PDX models. An integrative genomic classifier was developed using the random forest algorithm, trained on primary tumor data, and shown to identify PDX cancer subtypes with high accuracy. Citation Format: Zhengyan Kan, Edward Rosfjord, James Hardwick, Ying Ding, Xianxian Zheng, Julio Fernandez, Stephanie Shi, Mark Ozeck, Hui Wang, Gabriel Troche, Eric Upeslacis, Amy Jackson-Fisher, Keith Ching, Shibing Deng, Xie Tao, John Chionis, Maruja Lira, Xiaorong Li, Konstantinos Tsaparikos, Patrick Lappin, Pamela Vizcarra, David Shields, Judy Lucas, Paul Rejto. Molecular profiling of patient-derived xenograft models across cancers. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-33.

Breast cancers (BC) in younger, premenopausal patients (YBC) tend to be more aggressive with worse prognosis, higher chance of relapse and poorer response to endocrine therapies compared to breast cancers in older patients. The proportion of YBC (age ≤ 40) among BC in East Asia is estimated to be 16-32%, significantly higher than the 7% reported in Western countries. To characterize the molecular bases of Asian YBC, we have performed whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) on tumor and matched normal samples from 134 Korean BC patients consisting of 74 YBC cases (age ≤ 40) and 60 OBC cases (age > 40). We then performed comparison analyses and integrative analyses with the TCGA BC cohort consisting of 1,116 tumors from primarily Caucasian patients, also grouped by age into YBC (age ≤ 40), IBC (40 < age ≤ 60) and OBC (age > 60). Somatic mutation prevalence analysis identified 7 significantly mutated genes and the same top three genes – TP53, GATA3 and PIK3CA – were reported by the TCGA BC study. To identify differentially expressed (DE) genes and pathways in YBCs vs. OBCs, we performed logistic regression analyses while controlling for the confounding effects of tumor purity and stage. We were surprised to see a significant overlap in DE pathways between a comparison of adjacent normal tissues in younger vs. older TCGA cohorts and a comparison of YBC vs. OBC tumors, indicating that normal tissue compartment could contribute to observed differences between bulk tumors. To separately examine molecular signatures from tumor, stroma and normal compartments, we used non-negative matrix factorization (NMF) analyses to virtually dissect bulk tumor expression data and identified 14 factors including 3 factors associated with normal tissues, 1 factor associated with stroma and 1 factor associated with tumor infiltrating lymphocytes (TIL). Integrative analyses of tumor associated factors and DE pathways revealed that estrogen response, endocrine therapy resistance, and oxidative phosphorylation pathways are up-regulated in YBCs compared to OBCs while cell cycle and proliferation pathways are up-regulated in Asian OBCs. Interestingly, many immune and inflammation pathways correlated with the TIL factor were significantly upregulated in OBCs vs. YBCs. Using gene expression signatures representing distinct immune cell types, we classified our cohort into four subtypes of varying TIL activities and observed significant enrichment of the TIL-high subtype in OBCs compared to YBCs. These observations were confirmed by IHC analyses of four TIL markers (CD45, CD4, CD8 and CD163) in 120 tumors. To our knowledge, this is the first large-scale multi-omics study of Asian breast cancer and would significantly contribute to the compendium of molecular data available for studying young breast cancers. The major landmarks in the molecular landscape looked similar across BCs of different ethnicities and ages, however, we have identified a number of distinguishing molecular characteristics associated with Asian YBC. The sources for some signatures were further traced to non-tumor intrinsic compartments, indicating that tumor microenvironment may play potentially important roles in driving the carcinogenesis of young breast cancers. Citation Format: Kan Z, Ding Y, Cho S, Lee S-H, Powell E, Jung HH, Chung W, Deng S, Choi Y-l, Kim J, Park W-Y, Vizcarra P, Fernandez-Banet J, Nichols T, Ram S, Lee SK, Kim SW, Lee JE, Ching KA, Kim J-Y, Ahn JS, Im Y-H, Nam SJ, Park YH. Multi-omics and immuno-oncology profiling reveal distinct molecular signatures of young Asian breast cancers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-15.

Chronic hypoxia is a common inducer of end-stage cardiovascular disease. In cells under hypoxia, the hypoxia-inducible factor-1 (HIF-1) plays a vital role in regulating downstream target genes. However, the mechanism of hypoxia in cardiomyocytes is still unclear. In this study, we aimed to identify novel downstream epigenetic targets of HIF-1α in cardiomyocytes under hypoxia. H9c2 cells were exposed to hypoxia condition, and quantitative real-time PCR analysis was performed to evaluate the expression of miR-20b-5p. The results indicated that the expression of miR-20b-5p was down-regulated in H9c2 cells under low oxygen condition. Meanwhile, HIF-1α overexpression further down-regulated the miR-20b-5p expression in H9c2 cells transfected with HIF-1α plasmids. In addition, Annexin-V-FITC/PI flow cytometry analysis suggested that overexpression of miR-20b-5p attenuated cell apoptosis under hypoxia condition in H9c2 cells. Western blot analysis showed that the hypoxia apparently increased Bax and cleaved-caspase-3, but decreased Bcl-2 expression in H9c2 cells, indicating that hypoxia-induced NF-κB signaling pathway activation is mediated by miR-20b-5p. Hypoxia-induced H9c2 cell apoptosis was reduced after HIF-1α knockdown as shown by the flow cytometry analysis. In conclusion, we identified that miR-20b-5p plays an important role in mediating cardiomyocytes apoptosis under hypoxia, which is mediated by the HIF-1/NF-κB signaling pathway.

In this phase I study (NCT01307267), we evaluated safety, pharmacokinetics, clinical activity, and pharmacodynamics of treatment with utomilumab plus rituximab in patients with relapsed/refractory follicular lymphoma (FL) and other CD20Primary objectives were to assess treatment safety and tolerability for estimating the MTD, using a modified time-to-event continual reassessment method, and selecting the recommended phase II dose (RP2D).Sixty-seven patients received utomilumab (0.03-10.0 mg/kg every 4 weeks) and rituximab (375 mg/mUtomilumab in combination with rituximab demonstrated clinical activity and a favorable safety profile in patients with CD20

The applications of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in tissue engineering have been widely studied. This study aimed to compare the biocompatibility and osteoinductivity of single-walled carbon nanotubes (SWCNTs)/PHBV composites with multi-walled CNTs (MWCNTs)/PHBV composites. CNTs were dispersed in PHBV by ultrasonication and composites were created using thermal injection moulding. In order to test their biocompatibility and osteoinductivity. Rat osteoblasts (rOBs) were then cultured and seeded on the composites. The composites were implanted in rat femoral bone defects. Our results showed that lower weight percentages of SWCNTs and MWCNTs (2-4%) improved both their mechanical and thermal decomposition properties. However, further reduction of rOBs cell death was observed in MWCNTs/PHBV. SWCNTs were shown to upregulate the expression of Runx-2 and Bmp-2 in early stage significantly, while MWCNTs showed a stronger long-term effect on Opn and Ocn. The in vivo result was that MWCNTs/PHBV composites induced intact rounding new bone, increased integration with new bone, and earlier completed bone remodeling when compared with SWCNTs. Immunohistochemistry also detected higher expression of RUNX-2 around MWCNTs/PHBV composites. In conclusion, there were no differences observed between SWCNTs and MWCNTs in the reinforcement of PHBV, while MWCNTs/PHBV composites showed better biocompatibility and osteoinductivity both in vitro and in vivo.

Background: Percutaneous coronary intervention with the new generation drug eluting stents (DES) is 1 among the revascularization procedures required to treat patients with coronary artery disease (CAD). Since late stent thrombosis and silent myocardial infarction are highly associated with type 2 diabetes mellitus (T2DM), an analysis comparing the newer generation DES in this specific subgroup of patients would be scientifically relevant. In this analysis, we aimed to systematically compare the cardiovascular outcomes observed with the ultrathin bioresorbable polymer sirolimus eluting stents (SES) versus thin, durable polymer everolimus eluting stents (EES) following percutaneous coronary intervention in patients with T2DM. Methods: Through online databases, relevant studies comparing ultrathin bioresorbable polymer SES versus the durable polymer EES were carefully searched. The cardiovascular outcomes were assessed during a follow-up time period of 1 year and more than 1 year (1–5 years) respectively. This meta-analysis was carried out by the latest version of the RevMan software. Following analysis, the results were represented by odds ratios (OR) with 95% confidence intervals (CI). Results: A total number of 1967 patients with T2DM were included in this analysis. During a 1 year follow-up time period, target lesion failure (TLF) (OR: 0.59, 95% CI: 0.34–1.02; P = .06, target vessel revascularization (TVR) (OR: 0.97, 95% CI: 0.55–1.70; P = .91) and target lesion revascularization (TLR) (OR: 0.91, 95% CI: 0.44–1.87; P = .79) were similarly observed with ultrathin bioresorbable polymer SES versus the thin, durable polymer EES in these patients with T2DM. Other cardiovascular outcomes including myocardial infarction (MI), major adverse cardiac events, all-cause mortality (OR: 0.72, 95% CI: 0.37–1.40; P = .34), cardiac death and stent thrombosis (OR: 0.85, 95% CI: 0.45–1.62; P = .63) were also similarly observed with these 2 types of new stents. During a follow-up time period above 1 year (1–5 years), still no significant difference was observed in TLF, TVR, TLR, major adverse cardiac events, MI, all-cause mortality, cardiac death and stent thrombosis (OR: 0.62, 95% CI: 0.33–1.16; P = .14). Conclusions: The ultrathin bioresorbable polymer SES were similar to the durable polymer EES in these patients with T2DM. These 2 types of new generation stents were comparable in terms of cardiovascular outcomes. Hence, they might be recommended in patients with T2DM. Upcoming trials should be able to confirm this hypothesis.

The molecular bases underlying neoadjuvant chemotherapy (NAC) response are poorly understood. To elucidate the effects of NAC on breast tumor biology and its association with clinical outcome, we have conducted WES and RNA-Seq profiling of a longitudinal breast cancer (BC) cohort consisting of 146 cases (281 tumors, 109 pairs), including 55 (38%) that achieved pathologic complete responses (pCR) and 91 (62%) that harbored residual diseases at time of surgery. Tumor biopsies were collected for each patient at three time points - pre-treatment, three weeks after the first cycle of anthracycline and cyclophosphamide (AC) and at the time of surgery, after 3 more cycles of AC followed by 4 cycles of taxane. In addition to somatic mutations and copy number alterations, we also derived a comprehensive set of genomic and molecular features for each tumor including chromosomal instability, loss-of-heterozygosity, mutation burden, mutation signatures and expression signatures for oncogenic signaling pathways and immune cell subsets. Virtual microdissection analysis inferred 14 factors that represent distinct tissue compartment including a tumor infiltrating lymphocyte (TIL) factor and revealed that initial NAC treatment increased stromal and adjacent normal tissue fractions while reducing tumor cellularity. NAC also induced dynamic changes in immune gene expressions over time, a pattern that was validated through detecting and quantifying the density of TILs from H&E images. To investigate NAC induced changes in tumor intrinsic biology we classified tumors into five oncogenic cellular states on a reference Onco-GPS map defined by transcriptional signatures from breast cancer cell lines. We observed that, as a result of NAC treatment, tumors often change from one oncogenic state to another, transiently upregulating an EMT program that appears to mediate drug resistance and increase the likelihood of residual disease. Multiple regression and multivariate analyses were performed to identify predictive biomarkers of pCR status while adjusting for BC subtypes and tumor purity. We found that ER+ subtype and estrogen response signature but not Ki-67 were independently associated with NAC response. Within TNBC, the immunomodulatory subtype was enriched in responders while the basal-like subtype had the poorest response. Pretreatment TIL and changes in TIL level over time were independently associated with NAC response, implicating anti-tumor immunity in mediating the efficacy of chemotherapies. Through multi-omics characterization of longitudinally paired tumor biopsies, we have revealed dynamic changes in the tumor molecular states in BC patients undergoing NAC treatment, identified molecular markers of treatment outcome and derived insights into the mechanism of action as well as resistance to an important class of therapy. Citation Format: Samir Lal, Ying Ding, Jeong Eon Lee, Soo-Hyeon Lee, Se Kyung Lee, Jae-Yong Nam, Jong Han Yu, Yoon-la Choi, Seok Won Kim, Seok Jin Nam, Ji-Yeon Kim, Sripad Ram, Eric Powell, Keith A. Ching, Pablo Tamayo, William Kim, Huwate Yeerna, Soo Youn Cho, Vinicius Bonato, Shibing Deng, Jinho Kim, Hyuntae Shin, Woong-Yang Park, Paul A. Rejto, Jadwiga Bienkowska, Yeon-Hee Park, Zhengyan Kan. Multi-omics profiling of breast cancers during neoadjuvant chemotherapy identified distinct molecular changes and biomarkers associated with clinical response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1684.

Background: PF-06647020, an ADC comprising a humanized monoclonal antibody against PTK7, a cleavable valine-citrulline linker, and an auristatin payload, is being investigated in an ongoing Phase I clinical trial in pts with advanced solid tumors. We hypothesized that response to a PTK7-directed ADC would correlate with PTK7 expression in the pts tumor. We report preliminary baseline PTK7 expression in pts tumors and clinical response using a novel CLIA validated laboratory developed test (LDT). Methods: We developed and validated an LDT comprising an anti-PTK7 immunohistochemistry assay with digital tissue analysis using Flagship Biosciences Inc’s cTA® platform to detect membrane-associated PTK7 in formalin-fixed paraffin embedded (FFPE) epithelial malignancies of ovary, breast and lung. Tumor samples were immunolabeled with the LDT and image analysis was applied to derive a digital H-score. Specifically the digital image annotation included desired regions of analysis (ROA) and excluded regions not of interest. The cTA® platform identified each cell in a given ROA and quantified an overall H-score for malignant epithelial cell membranes immunolabeled for PTK7. Non-small cell lung cancer (NSCLC) pts with H-scores ≥ 56.8 were eligible for enrollment whereas ovarian cancer (OVCA) pts were enrolled unselected. Triple negative breast cancer (TNBC) pts were initially enrolled unselected, but after review a requirement for H-scores ≥ 92.8 was implemented for enrollment. Results: PTK7 IHC was performed on baseline FFPE samples from 69 pts and digital H-scores were generated (Table 1). An additional 7 pt samples were not amenable to analysis with the cTA® platform. Conclusions: A novel CLIA validated LDT was developed and implemented to assess PTK7 baseline levels in FFPE tumors from pts treated with PF-06647020. Clinical responses tended to correlate with H-scores that were higher than the mean for each tumor type. Table 1.PTK7 digital H-scores in Phase I dose expansionNumber of SamplesMinimum H-scoreMaximum H-scoreMean H-scoreMean H-score of Non-respondersMean H-score of RespondersOVCA31120089.280.2111.2NSCLC1993287167.8159.2200.3TNBC1953258159.2147.0224.3 Citation Format: Amy Jackson-Fisher, Navi Mehra, Roberto Gianani, Pamela Whalen, Pamela Vizcarra, Shibing Deng, Stephen McComish, Xiaohua Xin, Eric L. Powell. Protein tyrosine kinase 7 (PTK7) biomarker analysis in patients (pts) treated with PF-06647020, a PTK7 antibody-drug conjugate (ADC), in a phase I dose expansion study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4035.

Let ?$G_w$? be a weighted graph. The inertia of ?$G_w$? is the triple ?$\mathrm{In} (G_w)=(i_+ (G_w), i_ - (G_w), i_0(G_w))$?, where ?$i _+ (G_w)$?, ?$i_- (G_w)$?, ?$i_0(G_w)$? are, respectively, the number of the positive, negative and zero eigenvalues of the adjacency matrix ?$A(G_w)$? of ?$G_w$? including their multiplicities. A simple ?$n$?-vertex connected graph is called a ?$(k - 1)$?-cyclic graph provided that its number of edges equals ?$n + k - 2$?. Let ?$\theta(r_1, r_2, \dots, r_k)_w$? be an ?$n$?-vertex simple weighted graph obtained from ?$k$? weighted paths ?$(P_{r_1})_w, ((P_{r_2})_w, \dots,(P_{r_k})_w$? by identifying their initial vertices and terminal vertices, respectively. Set ?$\Theta_ k: = \{\theta(r_1, r_2, \dots, r_k)_w \colon r_1 + r_2 + \dots + r_k = n + 2k - 2\}$?. The inertia of the weighted graph ?$\theta(r_1, r_2, \dots, r_k)_w$? is studied. Also, the weighted ?$(k - 1)$?-cyclic graphs that contain ?$\theta(r_1, r_2, \dots, r_k)_w$? as an induced subgraph are studied. We characterize those graphs among ?$\Theta_ k$? that have extreme inertia. The results generalize the corresponding results obtained in X.Z. Tan, B.L. Liu, The nullity of (k - 1)-cyclic graphs, Linear Algebra Appl. 438 (2013) 3144-3153 and G.H. Yu et al., The inertia of weighted unicyclic graphs, Linear Algebra Appl. 448 (2014) 130-152. Naj bo ?$G_w$? utežen graf. Vztrajnost grafa ?$G_w$? je urejena trojica ?$\mathrm{In} (G_w) = (i_+ (G_w), i_ - (G_w), i_0(G_w))$?, kjer so ?$i _+ (G_w)$?, ?$i_- (G_w)$?, ?$i_0(G_w)$? število pozitivnih, negativnih in ničelnih lastnih vrednosti sosednostne matrike ?$A(G_w)$? grafa ?$G_w$? upoštevaje njihove večkratnosti. Enostaven ?$n$?-vozliščni povezan graf se imenuje ?$(k -1)$?-cikličen graf, če je število njegovih povezav enako ?$n+k-2$?. Naj bo ?$\theta(r_1, r_2, \dots, r_k)_w$? ?$n$?-vozliščni enostaven utežen graf, ki ga dobimo iz ?$k$? uteženih poti ?$(P_{r_1})_w, ((P_{r_2})_w, \dots,(P_{r_k})_w$? z identificiranjem njihovih začetnih vozlišč ter njihovih končnih vozlišč. Vpeljimo ?$\Theta_ k: = \{\theta(r_1, r_2, \dots, r_k)_w \colon r_1 + r_2 + \dots + r_k = n + 2k - 2\}$?. Študiramo vztrajnost uteženega grafa ?$\theta(r_1, r_2, \dots, r_k)_w$?. Prav tako študiramo utežene ?$(k - 1)$?-ciklične grafe, ki vsebujejo ?$\theta(r_1, r_2, \dots, r_k)_w$? kot induciran podgraf. Karakteriziramo tiste grafe izmed ?$\Theta_ k$?, ki imajo ekstremno vztrajnost. Naši rezultati posplošujejo tiste, ki sta jih dobila Tan in Liu leta 2013 ter Yu in dr. leta 2014.

An unbiased scanning methodology using ultra high-performance liquid chromatography coupled with high-resolution mass spectrometry was used to bank data and plasma samples for comparing the data generated at different dates. This method was applied to bank the data generated earlier in animal samples and then to compare the exposure to metabolites in animal versus human for safety assessment. With neither authentic standards nor prior knowledge of the identities and structures of metabolites, full scans for precursor ions and all ion fragments (AIF) were employed with a generic gradient LC method to analyze plasma samples at positive and negative polarity, respectively. In a total of 22 tested drugs and metabolites, 21 analytes were detected using this unbiased scanning method except that naproxen was not detected due to low sensitivity at negative polarity and interference at positive polarity; and 4'- or 5-hydroxy diclofenac was not separated by a generic UPLC method. Statistical analysis of the peak area ratios of the analytes versus the internal standard in five repetitive analyses over approximately 1 year demonstrated that the analysis variation was significantly different from sample instability. The confidence limits for comparing the exposure using peak area ratio of metabolites in animal plasma versus human plasma measured over approximately 1 year apart were comparable to the analysis undertaken side by side on the same days. These statistical analysis results showed it was feasible to compare data generated at different dates with neither authentic standards nor prior knowledge of the analytes.

Aim This study aimed at exploring changes in YAP expression and their effect on periodontitis (PD) combined with traumatic occlusion (TO). Materials and methods BALB/cJ mice were used to establish a PD model by local administration of Porphyromonas gingivalis (P.g, ATCC 33277) and a TO model by occlusal elevation (OE) using composite resin bonding on the bilateral maxillary molar. The mouse fibroblast cell line (L929) and pre-osteoblast cell line (MC3T3-E1) were subjected to cyclic tensile/compressive stress and inflammatory stimuli (lipopolysaccharide from Escherichia coli) to verify in vivo results. Results Severe bone resorption was observed by microCT scanning in OE with P.g group, when compared to OE only and P.g only groups. Mechanical stress caused by OE activated the Hippo-YAP pathway in periodontal tissues and upregulated the expression of JNK/AP-1. OE with P.g further promoted the expression of YAP and JNK/AP1, leading to the upregulation of the JNK/AP-1 related inflammatory cytokines TNF-α and IL6. Similar results were obtained when osteoblasts were subjected to mechanical stress in vitro. Conclusions Our study demonstrated that periodontitis with TO caused severe inflammation-induced bone resorption. Activation of YAP and upregulation of JNK/AP-1 induced by TO potentially aggravated the symptoms of PD.

SummaryWe report the preclinical evaluation of PF-06463922, a potent and brain-penetrant ALK/ROS1 inhibitor. Compared with other clinically available ALK inhibitors, PF-06463922 displayed superior potency against all known clinically acquired ALK mutations, including the highly resistant G1202R mutant. Furthermore, PF-06463922 treatment led to regression of EML4-ALK-driven brain metastases, leading to prolonged mouse survival, in a superior manner. Finally, PF-06463922 demonstrated high selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 will be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors because of secondary ALK kinase domain mutations and/or brain metastases.

To investigate the plasma levels of amyloid beta (Aβ) and select inflammatory mediators in patients with various stages of AMD compared to that of age-matched controls, and discern a relationship to disease severity. Plasma samples were obtained from AMD subjects at various stages of disease—early (drusen only), geographic atrophy (GA), neovascular AMD (CNV)—and from controls of similar age without AMD. Samples were analyzed using a commercially available ELISA kit (sixteen cytokines) or LC/MS/MS (Aβ isotypes). Descriptive statistics were compiled on all analytes. Analysis of covariance (ANCOVA) was conducted to compare each analyte across AMD groups while adjusting for sex and age of the patients, and in comparison to the control group. Receiver operating characteristics plots were generated for the strongest predictor variables. Levels of alternative spliced CC3 proteins were significantly different between controls and CNV groups (p

Objectives Amongst the fourth generation of PHAs is bio-plasticpoly3-hydroxybutyrate4-hydroxybutyrate (P34HB); it is thus appropriate to perform novel research on its uses and applications. The main objective of this study was to determine whether electrospun P34HB fibres would accommodate viability, growth and differentiation of mouse adipose-derived stem cells (mASCs). Materials and methods In the present study, we looked at P34HB in two forms, electrospun P34HB fibres and P34HB film. Morphology of electrospun P34HB fibres and P34HB film were characterized using scanning electron microscopy, fluorescence microscopy and confocal laser scanning microscopy, after cell seeding. Cell adhesion, proliferation and cytotoxicity tests were conducted on both by MTT and CCK-8 assays, respectively. After being cultured with osteogenic induction, expression of adipogenic genes Runx2, OPN and OCN, were examined by real-time PCR. Results By scanning electron microscopy, light microscopy and confocal laser scanning microscopy, we observed that the mASCs grew well associated with the P34HB materials. After MTT and CCK-8 assay, we concluded that P34HB would, indeed, be a material suitable for further cell adhesion and proliferation studies. More importantly, we found that the P34HB matrices promoted expression of Runx2, OPN and OCN with osteogenic induction. Conclusions In this investigation, we can confirm that the electrospun P34HB fibres accommodated survival, proliferation and differentiation of mASCs, and we have been able to draw the conclusion that fibre scaffolds produced by the electrospinning process are promising for application of bone tissue engineering.

Approximately 7% of non-small cell lung carcinomas (NSCLCs) harbor oncogenic fusions involving ALK, ROS1, and RET. Although tumors harboring ALK fusions are highly sensitive to crizotinib, emerging preclinical and clinical data demonstrate that patients with ROS1 or RET fusions may also benefit from inhibitors targeting these kinases. Using a transcript-based method, we designed a combination of 3' overexpression and fusion-specific detection strategies to detect ALK, ROS1 and RET fusion transcripts in NSCLC tumors. We validated the assay in 295 NSCLC specimens and showed that the assay is highly sensitive and specific. ALK results were 100% concordant with fluorescence in situ hybridization (FISH) (n = 52) and 97.8% concordant with IHC (n = 179) [sensitivity, 96.8% (95% CI 91.0%-98.9%); specificity, 98.8% (95% CI 93.6%-99.8%)]. For ROS1 and RET, we also observed 100% concordance with FISH (n = 46 and n = 15, respectively). We identified seven ROS1 and 14 RET fusion-positive tumors and confirmed the fusion status by RT-PCR and FISH. One RET fusion involved a novel partner, cutlike homeobox 1 gene (CUX1), yielding an in-frame CUX1-RET fusion. ROS1 and RET fusions were significantly enriched in tumors without KRAS/EGFR/ALK alterations. ALK/ROS1/RET/EGFR/KRAS alterations were mutually exclusive. As a single-tube assay, this test shows promise as a more practical and cost-effective screening modality for detecting rare but targetable fusions in NSCLC.

Current methods to quantify T-cell clonal expansion only account for variance due to random sampling from a highly diverse repertoire space. We propose a beta-binomial model to incorporate time-dependent variance into the assessment of differentially abundant T-cell clones, identified by unique T Cell Receptor (TCR) β-chain rearrangements, and show that this model improves specificity for detecting clinically relevant clonal expansion. Using blood samples from ten healthy donors, we modeled the variance of T-cell clones within each subject over time and calibrated the dispersion parameters of the beta distribution to fit this variance. As a validation, we compared pre- versus post-treatment blood samples from urothelial cancer patients treated with atezolizumab, where clonal expansion (quantified by the earlier binomial model) was previously reported to correlate with benefit. The beta-binomial model significantly reduced the false-positive rate for detecting differentially abundant clones over time compared to the earlier binomial method. In the urothelial cancer cohort, the beta-binomial model enriched for tumor infiltrating lymphocytes among the clones detected as expanding in the peripheral blood in response to therapy compared to the binomial model and improved the overall correlation with clinical benefit. Incorporating time-dependent variance into the statistical framework for measuring differentially abundant T-cell clones improves the model's specificity for T-cells that correlate more strongly with the disease and treatment setting of-interest. Reducing background-level clonal expansion, therefore, improves the quality of clonal expansion as a biomarker for assessing the T cell immune response and correlations with clinical measures.

Background: CDK4/6 inhibition combined with endocrine therapy (ET) is now the standard of care for advanced hormone receptor (HR) positive breast cancer patients (pts). Previous ctDNA analysis of paired pre-treatment (pre-T) and end of treatment (EoT) plasma samples from the PALOMA-3 trial of the CDK4/6 inhibitor P with/without F demonstrated that acquired PIK3CA, ESR1, RB1 and rare growth factor receptor mutations (mut) likely contribute to resistance (Turner et al, ASCO 2018, Abstract 1001). We now report results from an extended ctDNA analysis of paired PALOMA-3 plasma samples using hybridization capture-based, next-generation sequencing (NGS) assay covering 87 breast cancer, HR signaling, and cell cycle-related genes. Methods: Pre-/post-menopausal pts whose disease progressed after prior ET (N=521) were randomized 2:1 to receive F 500 mg + P (125 mg/d, 3 wk on/1 wk off) or placebo. Cell-free DNA extracted from paired plasma samples (n=194) was analyzed with a custom 87-gene NGS assay, to identify single nucleotide variants and copy number amplification (CNA), with molecular barcoding, high NGS coverage (10,000-20,000 reads per nucleotide position), and background error correction. Differences in ctDNA mut frequencies detected in P+F compared to F alone were assessed and their association to clinical outcome estimated. Results: Blinded assay qualification and droplet digital PCR validation suggested a mut detection frequency cutoff at 0.3%. A high coefficient of correlation with previously presented data was observed for ESR1 and PIK3CA variants (r=0.94 and 0.97, respectively), with acquisition of PIK3CA and ESR1 mutations at EoT in P+F and F groups. Gene level mut analysis of EoT plasma revealed no significant difference between P+F vs F alone (Table), although there was an increase in RB1 mutations in P+F consistent with previous data. Other acquired muts at EoT (SMAD4, NOTCH2, and CDKN1B) were observed at low frequencies. Muts in CDK4 and CDK6 were rarely observed (< 1% of pts), regardless of treatment arm. Detected Variants, Frequency, n (%) P+FF GenePre-TEoTPre-TEoTPre-T pvalEoT pvalPIK3CA47(37)51(40)19(28)22(33)0.2660.352ESR136(28)45(35)19(28)24(36)11TP5330(24)33(26)23(34)25(37)0.1280.137RB12(2)9(7)2(3)2(3)NA0.336PTEN5(4)7(6)3(4)3(4)11AKT17(6)7(6)2(3)2(3)0.7210.721 Conclusions: Our results corroborate the previously reported results demonstrating that genomic profiles of treatment resistant cancer are similar between P + F and F therapy. Expanded analysis of cell-cycle genes identified no new recurrently mutated genes, and confirmed that RB1 mutations are selected at low frequency after P+F treatment. Alterations in cell cycle genes may not be a common mechanism of resistance to CDK4/6 inhibitors in HR+ HER2- advanced breast cancer. Sponsor: Pfizer Citation Format: O'Leary B, Lira ME, Huang S, Deng S, Xie T, Kinong JK, Hardwick J, Rejto P, Liu Y, Huang X, Huang Bartlett C, André F, Loibl S, DeMichele A, Cristofanilli M, Turner NC. Longitudinal ctDNA sequencing using an expanded genomic panel in the PALOMA3 trial of palbociclib plus fulvestrant [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD2-02.

// Seung Tae Kim 1, * , Maruja Lira 2, * , Shibing Deng 2 , Sujin Lee 1 , Young Suk Park 1 , Ho Yeong Lim 1 , Won Ki Kang 1 , Mao Mao 3 , Jin Seok Heo 4 , Wooil Kwon 4 , Kee‑Taek Jang 5 , Jeeyun Lee 1, 6 , Joon Oh Park 1, 6 1 Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 2 Precision Medicine, Oncology Research Unit, Pfizer, Inc, San Diego, CA, USA 3 WuXi AppTec, Shanghai, China 4 Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 5 Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 6 Innovative Cancer Medicine Institute, Samsung Medical Center, Seoul, Korea * These authors have contributed equally to this work Correspondence to: Joon Oh Park, e-mail: oncopark@skku.edu Jeeyun Lee, e-mail: jyunlee@skku.edu Keywords: cell free DNA (cfDNA), PIK3CA mutation, droplet digital PCR (ddPCR) Received: July 13, 2015 Accepted: October 05, 2015 Published: October 16, 2015 ABSTRACT PIK3CA mutation is considered a good candidate for targeted therapies in cancers, especially biliary tract cancer (BTC). We evaluated the utility of cell free DNA (cfDNA) from serum by using droplet digital PCR (ddPCR) as an alternative source for PIK3CA mutation analysis. To identify matching archival tumour specimens from serum samples of advanced BTC patients, mutation detection using ddPCR with Bio-Rad’s PrimePCR mutation and wild type assays were performed for PIK3CA p.E542K, p.E545K, and p.H1047R. Thirty-eight patients with metastatic BTC were enrolled. Only one (BTC 29T) sample ( n = 38) was positive for PIK3CA p.E542K and another (BTC 27T) for p.H1047R mutation; none was positive for PIK3CA p.E545K. Matched serum sample (BTC 29P) was positive for PIK3CA p.E542K with 28 mutant copies detected, corresponding to 48 copies/ml of serum and an allelic prevalence of 0.3%. Another matched serum sample (BTC 27P) was positive for PIK3CA p.H1047R with 10 mutant copies detected, i.e. 18 copies/ml and an allelic frequency of 0.2%. High correlation was noted in the PIK3CA mutation status between tumour gDNA and serum cfDNA. Low-level PIK3CA mutations were detectable in the serum indicating the utility of cfDNA as a DNA source to detect cancer-derived mutations in metastatic biliary cancers.

Background Progression of hepatocellular carcinoma (HCC) often leads to vascular invasion and intrahepatic metastasis, which correlate with recurrence after surgical treatment and poor prognosis. The molecular prognostic model that could be applied to the HCC patient population in general is needed for effectively predicting disease-free survival (DFS).

Breast cancers (BC) in younger, premenopausal patients (YBC) tend to be more aggressive with worse prognosis, higher chance of relapse and poorer response to endocrine therapies compared to breast cancers in older patients. The proportion of YBC (age ≤ 40) among BC in East Asia is estimated to be 16-32%, significantly higher than the 7% reported in Western countries. In addition, approximately half of the Asian BC patients were premenopausal compared to 15-30% in the West. To characterize the molecular bases of Asian YBC, we have performed whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) on tumor and matched normal samples from 168 Korean BC patients consisting of 106 YBC cases (age ≤ 40) and 62 OBC cases (age > 40). We then performed comparison analyses with the TCGA BC cohort consisting of 1,116 tumors from primarily Caucasian patients, also grouped by age into YBC (age ≤ 40), IBC (40 < age ≤ 60) and OBC (age > 60). We performed logistic regression analyses to identify differentially expressed (DE) genes and pathways among age-based cohorts while controlling for the confounding effects of molecular subtype, tumor purity and stage. Within the Asian cohort, we found that estrogen response, endocrine therapy resistance, and various metabolism pathways are up-regulated in YBCs while cell cycle, proliferation and inflammatory pathways are up-regulated in OBCs. To separately examine molecular signatures from tumor, stroma and normal compartments, we used non-negative matrix factorization (NMF) analyses to virtually dissect bulk tumor expression data and identified 14 factors including 3 factors associated with normal tissues, 1 factor associated with stroma and 1 factor associated with tumor infiltrating leukocytes (TILs). By examining the correlation between pathway gene expression and NMF factors, we inferred that DE pathways such as fatty acid metabolism, bile acid biosynthesis, and epithelial-to-mesenchymal transition (EMT) were mainly active in stromal and normal tissue compartments. The TIL factor was significantly enriched in Asian BCs relative to Caucasian BCs with the highest TIL factor weight observed in Asian OBCs. Using gene expression signatures representing distinct types of TILs, we classified the combined cohort into three subtypes of varying TIL activities. Consistent with results from the NMF analysis, the TIL-high subtype is also significantly enriched in Asian BCs relative to Caucasian BCs. To our knowledge, this is the first large-scale multi-omics study of Asian breast cancer. Comparative analyses of multi-omics profiles from Asian and primarily Caucasian BC cohorts identified distinguishing molecular signatures associated with Asian BCs. Further, many signatures appeared to be specific to non-tumor compartments within bulk tumor, indicating that young Asian BCs may harbor distinctive tumor microenvironment. Citation Format: Yeon Hee Park, Ying Ding, Soo-Hyeon Lee, Hae Hyun Jung, Woosung Chung, Soonweng Cho, Jin-Ho Kim, Shibing Deng, Yoon-la Choi, Julio Fernandez, Se Kyung Lee, Seok Won Kim, Jeong Eon Lee, Ji-Yeon Kim, Jin Seok Ahn, Young-Hyuck Im, Seok Jin Nam, Woong-Yang Park, Zhengyan Kan. Comparative analyses of multi-omics profiles reveal distinctive molecular signatures of young Asian breast cancers [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 3370. doi:10.1158/1538-7445.AM2017-3370

Liquid biopsies have the potential to revolutionize the way physicians select personalized anti-cancer therapies, monitor patient responses to treatment, and characterize acquired resistance to cancer drugs. New tests that use a simple peripheral blood draw offer snapshots of a patient‘s total tumor DNA mutation profile and are attractive because of their minimally-invasive modality and because they integrate information from both primary and metastatic disease. Currently, most plasma cell-free DNA (cfDNA) mutation detection tests used in clinical research detect known hotspot mutations in a limited number of genes. Technologies that interrogate multi-gene panels in cfDNA are advancing, but commercially-available options suitable for clinical use are limited, come at a high cost, and are not customizable. We designed and developed a customized, next generation sequencing-based, liquid biopsy assay capable of detecting somatic mutations in 87 breast cancer genes involved in cell cycle and estrogen receptor signaling. Targeted regions (147 Kb) were enriched using hybrid capture resulting in an average capture specificity and uniformity of 65.93% and 96.38%, respectively. When tested on cfDNA from healthy donors (n=14), we demonstrated a level of specificity >99.99%. Analytical sensitivity of 0.1% was established on HapMap and reference mutant cell line DNA. Using a pool of HapMap genomic DNA (n=10), 96% (48/50) of SNPs with expected allele frequency of 0.1% were detected. In reference mutant cell line DNA with 1% or 0.1% mutant allele frequencies, we were able to reliably detect all mutations present at 1% and mutations at 0.1% in 50% of the cases. Assay validation on plasma cfDNA with matching tumor from ER+, HER2- breast cancer patients will be presented. In conclusion, we developed a highly sensitive and specific liquid biopsy assay to interrogate 87 breast cancer-related genes. The high level of specificity and sensitivity makes the test ideal not only for detecting known cancer gene hotspot mutations but also for detection of novel gene mutations that may arise during treatment as a result of acquired drug resistance. Citation Format: Maruja E. Lira, Tao Xie, Shibing Deng, Jennifer Kinong, Jingjin Gao, Zhou Zhu, Nathan Lee, Paul Rejto, Jadwiga Bienkowska, James Hardwick, Kai Wang, Stephen Huang. Liquid biopsy testing allows highly-sensitive detection of plasma cfDNA mutations in 87 breast cancer-related genes [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 2749. doi:10.1158/1538-7445.AM2017-2749

Schizophrenia is characterized by affective, cognitive, neuromorphological, and molecular abnormalities that may have a neurodevelopmental origin. MicroRNAs (miRNAs) are small noncoding RNA sequences critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of 854 miRNAs in prefrontal cortical tissue from 100 control, schizophrenic, and bipolar subjects. The cyclic AMP-responsive element binding- and NMDA-regulated microRNA miR-132 was significantly down-regulated in both the schizophrenic discovery cohort and a second, independent set of schizophrenic subjects. Analysis of miR-132 target gene expression in schizophrenia gene-expression microarrays identified 26 genes up-regulated in schizophrenia subjects. Consistent with NMDA-mediated hypofunction observed in schizophrenic subjects, administration of an NMDA antagonist to adult mice results in miR-132 down-regulation in the prefrontal cortex. Furthermore, miR-132 expression in the murine prefrontal cortex exhibits significant developmental regulation and overlaps with critical neurodevelopmental processes during adolescence. Adult prefrontal expression of miR-132 can be down-regulated by pharmacologic inhibition of NMDA receptor signaling during a brief postnatal period. Several key genes, including DNMT3A , GATA2 , and DPYSL3 , are regulated by miR-132 and exhibited altered expression either during normal neurodevelopment or in tissue from adult schizophrenic subjects. Our data suggest miR-132 dysregulation and subsequent abnormal expression of miR-132 target genes contribute to the neurodevelopmental and neuromorphological pathologies present in schizophrenia.

The genotoxicity testing battery is highly sensitive for detection of chemical carcinogens. However, it features a low specificity and provides only limited mechanistic information required for risk assessment of positive findings. This is especially important in case of positive findings in the in vitro chromosome damage assays, because chromosome damage may be also induced secondarily to cell death. An increasing body of evidence indicates that toxicogenomic analysis of cellular stress responses provides an insight into mechanisms of action of genotoxicants. To evaluate the utility of such a toxicogenomic analysis we evaluated gene expression profiles of TK6 cells treated with four model genotoxic agents using a targeted high density real-time PCR approach in a multilaboratory project coordinated by the Health and Environmental Sciences Institute Committee on the Application of Genomics in Mechanism-based Risk Assessment. We show that this gene profiling technology produced reproducible data across laboratories allowing us to conclude that expression analysis of a relevant gene set is capable of distinguishing compounds that cause DNA adducts or double strand breaks from those that interfere with mitotic spindle function or that cause chromosome damage as a consequence of cytotoxicity. Furthermore, our data suggest that the gene expression profiles at early time points are most likely to provide information relevant to mechanisms of genotoxic damage and that larger gene expression arrays will likely provide richer information for differentiating molecular mechanisms of action of genotoxicants. Although more compounds need to be tested to identify a robust molecular signature, this study confirms the potential of toxicogenomic analysis for investigation of genotoxic mechanisms.

Our group has been using the ECVAM Embryonic Stem Cell assay to predict developmental toxicity. In order to improve the separation of non-teratogens from weak teratogens, we have employed measures of gene expression, and different statistical methods from those originally used to develop the test. These approaches have fundamentally not improved the discrimination of 'weaks' from 'nons'. A realization that a very low value for cytotoxicity IC50 would drive a final result for the test in ways that were inappropriate for pharmaceuticals has led us to re-examine the cytotoxicity component. Our current efforts are focused on other, perhaps more sensitive, measures of cytotoxicity, combined with gene expression changes in mouse stem cells in an attempt to correctly identify weak teratogens and non-teratogens.

Breast cancers (BC) in younger, premenopausal patients (YBC) tend to be more aggressive with worse prognosis, higher chance of relapse and poorer response to endocrine therapies compared to breast cancers in older patients (OBC). The proportion of YBC (age ? 40) among BC in East Asia is estimated to be 16-32%, significantly higher than the 7% reported in Western countries. Genomic and molecular characterizations have deepened our understanding of breast cancer biology in areas ranging from intrinsic subtypes to treatment responses, however, the molecular bases of Asian YBC remains poorly characterized. We have performed whole-exome sequencing (WES), whole-transcriptome sequencing (WTS) and high coverage targeted sequencing on tumor and matched normal samples from 133 Korean BC patients consisting of 74 YBC cases (age ? 40). We further performed immunohistochemistry (IHC) analyses to characterize tumor-infiltrating lymphocytes (TILs) in 46 tumors using four markers (CD45, CD4, CD8 and CD163). We found that BRCA1/2 germline deleterious mutations are enriched in YBC and the ER+/HER2- subtype, indicating that Asian ER+ YBC has a significant germline contribution. MutSig analysis4 identified ARID1A as a significantly mutated gene, implicating chromatin modeling as a cancer driver in Asian BC. Differential expression analyses suggested that Asian YBC differ in energy metabolism and are more active in protein synthesis than OBC tumors, whereas OBC is more proliferative than YBC. Using gene expression signatures representing distinct immune cell types and immunohistochemistry, we classified our cohort into four subtypes of varying TIL activities: high, medium, low and quiet. The majority of immunogenic cases with high TIL levels lie in ER+ or HER2+ subtypes although higher proportion is seen in TNBC. Moreover, YBC tumors appear to harbor lower levels of TIL activities than OBC, suggesting that younger patients may be less likely to benefit from immunomodulatory therapies than older patients. To our knowledge, this is the first large-scale multi-omics study of Asian breast cancer and would significantly contribute to the compendium of molecular data available for young, premenopausal breast cancer. While the major landmarks in the molecular and immune landscape of Asian BC look similar to that of the predominantly Caucasian BC cohorts, we have identified a number of distinguishing characteristics pointing to distinctive oncogenic mechanisms underlying Asian BC. Citation Format: Yeon Hee Park, Ying Ding, Soonweng Cho, Soo-Hyeon Lee, Hae Hyun Jung, Woosung Chung, Jinho Kim, Woong-Yang Park, Eric Powell, Pamela Vizcarra, Shibing Deng, Se Kyung Lee, Seok Won Kim, Jeong Eon Lee, Ji-Yeon Kim, Jin Seok Ahn, Young-Hyuck Im, Seok Jin Nam, Zhengyan Kan. Multi-omics and immuno-oncology profiling of an Asian breast cancer cohort enriched in young and premenopausal patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-325.

One of the main challenges in immuno-oncology is to quantify different types of immune cells in the tumor microenvironment, as such data can greatly facilitate our understanding of the mechanism of action of, and response to cancer immunotherapies. Traditionally immune profiling is performed by immunohistochemistry or flow cytometry experiments using surface markers specific to each immune cell type. However, both approaches suffer from practical limitations such as reagent availability and real world specimen conditions. Recently several computational deconvolution approaches using expression profiles of the bulk tumor tissue samples have been developed. This alternative approach utilizes existing database of purified immune cell gene signatures and relies on a rigorous mathematical framework of signal deconvolution. One of such algorithms, called CIBERSORT (Newman et al. 2015), has been demonstrated to perform robustly in deconvoluting the relative fractions of 22 human leukocyte subsets in solid tumor tissue samples, and benchmarked favorably against FACS analysis. In this work, we performed immune phenotyping by the CIBERSORT-based expression deconvolution on a cohort of 100 gastric cancer (GC) patients. We found that the fractions of activated CD4 memory T cells, rather than CD8+ T cell, are most significantly correlated with tumor neo-antigen load, whereas the latter is most significantly associated with the expression of a “Cytolytic Activity” metagene and an interferon gamma signature. There is stronger pre-existing host immune response against the MSI and EBV subtypes of GCs mediated by either cytotoxic T cells or Natural Killer cells. Lastly, we found a high M2/M1 tumor associated Macrophages (TAMs) ratio is strongly associated with poor GC prognosis, corroborating recent reports on the role of a TAM and stromal response in gastric cancer angiogenesis. Taken together, we demonstrated in this work that critical information about the tumor infiltrating immune cells can be gleaned from the computational deconvolution of bulk tumor gene expression profiling data. The comprehensiveness and cost-effectiveness of this approach can complement other immune profiling techniques in characterizing a wide variety of tumor specimens under various treatment conditions. We foresee its application in characterizing pharmacodynamics, understanding immunological mechanism of action and monitoring treatment response and disease progression for cancer immunotherapies and their combinations in both pre-clinical and clinical settings. Citation Format: Kai Wang, Hoicheong Siu, Shibing Deng, Jadwiga R. Bienkowska, Suet Yi Leung. Characterizing the immunogenicity of gastric cancer by transcriptomic expression based immune phenotyping. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4143.

Consistency and coherence of gene expression data across multiple sites depends on several factors such as platform (oligo, cDNA, etc.), environmental conditions at each laboratory, and data quality. The Hepatotoxicity Working Group of the International Life Sciences Institute Health and Environmental Sciences Institute consortium on the application of genomics to mechanism-based risk assessment is investigating these factors by comparing high-density gene expression data sets generated on two sets of RNA from methapyrilene (MP) experiments conducted at Abbott Laboratories and Boehringer-Ingelheim Pharmaceuticals, Inc. using a single platform (Affymetrix Rat Genome U34A GeneChip) at seven different sites. This article focuses on the evaluation of data quality and statistical models that facilitate the comparison of such data sets at the probe level. We present methods for exploring and quantitatively assessing differences in the data, with the principal goal being the generation of lists of site-insensitive genes responsive to low and high doses of MP. A combination of numerical and graphical techniques reveals important patterns and partitions of variability in the data, including the magnitude of the site effects. Although the site effects are significantly large in the analysis results, they appear to be primarily additive and therefore can be adjusted in the statistical calculations in a way that does not bias conclusions regarding treatment differences.

Characteristic patterns of gene expression measured by DNA microarrays have been used to classify tumors into clinically relevant subgroups. In this study, we have refined the previously defined subtypes of breast tumors that could be distinguished by their distinct patterns of gene expression. A total of 115 malignant breast tumors were analyzed by hierarchical clustering based on patterns of expression of 534 “intrinsic” genes and shown to subdivide into one basal-like, one ERBB2 -overexpressing, two luminal-like, and one normal breast tissue-like subgroup. The genes used for classification were selected based on their similar expression levels between pairs of consecutive samples taken from the same tumor separated by 15 weeks of neoadjuvant treatment. Similar cluster analyses of two published, independent data sets representing different patient cohorts from different laboratories, uncovered some of the same breast cancer subtypes. In the one data set that included information on time to development of distant metastasis, subtypes were associated with significant differences in this clinical feature. By including a group of tumors from BRCA1 carriers in the analysis, we found that this genotype predisposes to the basal tumor subtype. Our results strongly support the idea that many of these breast tumor subtypes represent biologically distinct disease entities.

This study evaluated factors that contribute to the increased energy cost of locomotion in youth. The subjects were 321 8-18-year-old youth, similar dispersed by age and sex. Oxygen uptake (VO2) was measured during rest (REE), running at 8 km · h−1 and cycling at 16 km · h−1, using a COSMED K4b2 metabolic system. Developmental stage was obtained via questionnaire. Stature, body mass, and skinfolds (triceps & subscapular) were measured. Both sexes had similar absolute VO2 (mL · min−1) at rest (p = 0.065) and running (p = 0.084), but the males had a higher VO2 during cycling (p = 0.046). There were no sex differences in relative VO2 (mL · kg−1 · min−1) at rest (p = 0.083); however, the males had a higher VO2 than the females during cycling and running (p £ 0.002). Multiple regression, tested for collinearity, found that absolute VO2 during cycling and running was mostly related to fat-free mass (p = 0.0001). Similar analyses for relative VO2 (mL · kg−1 · min−1) during cycling found that fat-free mass, sex, and skinfolds were significant contributors (p ‡ 0.003). During running the relative VO2 was related to skinfolds, fat-free mass, and resting energy expenditure (p < 0.05). Neither age nor developmental stage was a significant contributor. The results indicate that the VO2 of locomotion is most closely associated with fat-free mass. Thus, to compare youth of varying age or pubertal developmental status, fat-free mass should be taken into consideration.