Biomarker Of Disease Burden Research Articles

Quantitative reliability assessment of brain MRI volumetric measurements in type II GM1 gangliosidosis patients.

GM1-gangliosidosis (GM1) leads to extensive neurodegenerative changes and atrophy that precludes the use of automated MRI segmentation techniques for generating brain volumetrics. We developed a standardized segmentation protocol for brain MRIs of patients with type II GM1 and then assessed the inter- and intra-rater reliability of this methodology. The volumetric data may be used as a biomarker of disease burden and progression, and standardized methodology may support research into the natural history of the disease which is currently lacking in the literature. Twenty-five brain MRIs were included in this study from 22 type II GM1 patients of which 8 were late-infantile subtype and 14 were juvenile subtype. The following structures were segmented by two rating teams on a slice-by-slice basis: whole brain, ventricles, cerebellum, lentiform nucleus, thalamus, corpus callosum, and caudate nucleus. The inter- and intra-rater reliability of the segmentation method was assessed with an intraclass correlation coefficient as well as Sorensen-Dice and Jaccard coefficients. Based on the Sorensen-Dice and Jaccard coefficients, the inter- and intra-rater reliability of the segmentation method was significantly better for the juvenile patients compared to late-infantile (p < 0.01). In addition, the agreement between the two rater teams and within themselves can be considered good with all p-values < 0.05. The standardized segmentation approach described here has good inter- and intra-rater reliability and may provide greater accuracy and reproducibility for neuromorphological studies in this group of patients and help to further expand our understanding of the natural history of this disease.

Elenestinib, an Investigational, Next Generation KIT D816V Inhibitor, Reduces Mast Cell Burden, Improves Symptoms, and Has a Favorable Safety Profile in Patients with Indolent Systemic Mastocytosis: Analysis of the Harbor Trial

Introduction Indolent systemic mastocytosis (ISM) is a rare clonal mast cell disease driven by the KIT D816V mutation in nearly all patients. Patients with ISM can present with debilitating acute and chronic cutaneous, gastrointestinal, neurocognitive, and systemic symptoms caused by the release of inflammatory mediators from abnormal mast cells. In some patients, symptoms are not adequately controlled with best supportive care (BSC) medications. Elenestinib (BLU-263) is a novel, investigational, oral, next-generation tyrosine kinase inhibitor that potently and selectively inhibits KIT D816V with limited central nervous system penetration and pharmacokinetics (PK) that support once daily dosing. HARBOR (NCT04910685) is a randomized, double-blind, placebo-controlled, phase 2/3 study assessing efficacy and safety of elenestinib in patients with ISM. Methods Eligible adult patients with ISM, confirmed following central review of bone marrow (BM) pathology, clinical, and laboratory findings per World Health Organization criteria and a moderate-to-severe symptom score based on minimum mean total symptom score (TSS) of the ISM-Symptom Assessment Form (ISM-SAF©2018), were stratified by baseline serum tryptase (&amp;lt;20 ng/mL vs ≥20 ng/mL) and randomly assigned 3:1 to elenestinib 25 mg, 50 mg, or 100 mg once daily + BSC (“elenestinib”) or placebo + BSC (“placebo”). The primary objective of Part 1 was to determine the recommended dose based on safety, PK, and pharmacodynamics. Secondary endpoints included changes in biomarkers of disease burden (serum tryptase, KIT D816V variant allele fraction [VAF], and BM mast cells) and ISM-SAF TSS. Three additional open-label PK groups enrolled patients at 50 mg, 75 mg, and 100 mg to further characterize PK and safety. Results As of data cutoff on July 3, 2023, a total of 122 patients with ISM have received their assigned treatment in Part 1 of the study; 39 were blinded and randomized to elenestinib or placebo, and 83 were treated with open-label elenestinib in the PK groups (21 patients at 50 mg, 34 patients at 75 mg, and 28 patients at 100 mg). Baseline demographics were similar to those reported for the general ISM population. After 12 weeks of therapy, symptom improvement was observed for all dose cohorts, and symptom reduction by TSS was greater for patients on elenestinib versus placebo in the blinded portion of Part 1. Patients receiving elenestinib at 25 mg, 50 mg, and 100 mg doses showed reduction (mean percent) from baseline for tryptase (-15.4%, -50.9%, and -68.4% vs 3.3%, respectively) and KIT D816VVAF (-37.5%, -70.3%, and -77.0% vs -2.5%, respectively) as compared to placebo. Similar reductions in TSS and disease-related biomarkers were observed in the open-label PK cohorts. After a median treatment duration of 35.3 weeks, elenestinib was well-tolerated at all dose levels. There were no treatment-related serious adverse events and no treatment-related adverse events that led to drug discontinuation. Conclusion Elenestinib at all tested doses demonstrated beneficial effects on disease-related symptoms and biomarkers of mast cell burden in this large and maturing cohort of patients with ISM with a moderate-to-severe symptom burden. Elenestinib was well tolerated across all dose levels with a promising early benefit-risk profile in ISM. These preliminary findings will inform dosing for the planned Part 2 of HARBOR, which will examine elenestinib versus placebo in a randomized, blinded, placebo-controlled setting.

Abstract 5596: Cell free DNA levels and fragmentation patterns in different liquid biopsy analytes (blood, urine and vaginal fluid) in cervical cancer patients

Abstract Background: Cervical cancer (CC) is the 4th most commonly diagnosed cancer among women, with approximately 528,000 new cases annually. Current screening approaches for this disease have certain limitations; Pap tests require dedicated cytopathology infrastructure, while human papillomavirus (HPV) DNA testing may lead to invasive procedures in patients with transient infection. Liquid biopsy has emerged as a minimally invasive approach to detect and monitor disease progression and treatment response. We and others have previously demonstrated the clinical utility of circulating tumor (ct)DNA to monitor HPV+ cancers. Moreover, cell free (cf)DNA fragmentation patterns have been found to be a biomarker of disease burden. This cfDNA is thought to originate largely as a result of cell death, where small fragments of ~167 bp are associated with apoptosis and larger fragments (&amp;gt;1000 bp) are associated with necrosis. Despite advances in liquid biopsy techniques, little is known about the composition of cfDNA from different analytes in CC patients. Methods: The aim of this study was to compare the presence and composition of cfDNA from different liquid biopsy analytes in patients with CC and high grade cervical intraepithelial neoplasia or dysplasia (CIN/CD). Blood, urine, and vaginal swabs were collected from 20 patients with CC and 9 patients with CIN/CD at the McGill University Health Centre. All samples were centrifuged twice to isolate supernatant. Samples were tested for HPV ctDNA by ddPCR. Analysis of fragment length was performed using the Agilent Bioanalyzer 2100. Dominant fragment was determined as the DNA fragment with the highest concentration, while overall fragment was calculated as the average fragment across all bioanalyzer peaks. Results: HPV16/18 ctDNA was detectable 14/20 CC patients and 2/6 CIN patients, and 0/3 CD patients in plasma, urine, and vaginal swab. Concordance for all sample types tested was seen in 90% of cases. On average, fragment analysis demonstrated 174, 195, and 183 bp dominant small fragments in plasma, urine and vaginal swab, respectively. Plasma samples showed only smaller fragments (range: 168-187 bp). Other analytes displayed a predominance of larger cfDNA, with an overall fragment size of 3996 and 5194 bp in urine and vaginal swab, respectively. Conclusions: HPV-DNA was detectable in all analytes sampled in patients with CC and CIN, with a trend of higher detection in CC samples. The fragmentation patterns of cfDNA varied between patients and within patients across analytes, with larger fragments - likely of necrotic origin - seen only in urine and vaginal swab cfDNA. Smaller fragments - likely related to apoptosis - were seen across all sample types studied. Overall, analysis of fragmentation may provide valuable insight into cfDNA origins in different sample types and provide a novel biomarker for diagnosis and surveillance. Citation Format: Sarah Tadhg Ferrier, Erica Mandato, Alexandra Bartolomucci, Thupten Tsering, Shuk On Annie Leung, Julia Valdemarin Burnier. Cell free DNA levels and fragmentation patterns in different liquid biopsy analytes (blood, urine and vaginal fluid) in cervical cancer patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5596.

Normal ex vivo mesenchymal stem cell function combined with abnormal immune profiles sets the stage for informative cell therapy trials in idiopathic pulmonary fibrosis patients

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic, progressive pulmonary disease characterized by aberrant tissue remodeling, formation of scar tissue within the lungs and continuous loss of lung function. The areas of fibrosis seen in lungs of IPF patients share many features with normal aging lung including cellular senescence. The contribution of the immune system to the etiology of IPF remains poorly understood. Evidence obtained from animal models and human studies suggests that innate and adaptive immune processes can orchestrate existing fibrotic responses. Currently, there is only modest effective pharmacotherapy for IPF. Mesenchymal stem cells (MSCs)-based therapies have emerged as a potential option treatment of IPF. This study characterizes the functionality of autologous MSCs for use as an IPF therapy and presents an attempt to determine whether the disease occurring in the lungs is associated with an alterated immune system.MethodsComprehensive characterization of autologous adipose-derived MSCs (aMSCs) from 5 IPF patient and 5 age- and gender-matched healthy controls (HC) was done using flow cytometry, PCR (ddPCR), multiplex Luminex xMAP technology, confocal microscopy self-renewal capacity and osteogenic differentiation. Additionally, multi-parameter quantitative flow cytometry of unmanipulated whole blood of 15 IPF patients and 87 (30 age- and gender-matched) HC was used to analyze 110 peripheral phenotypes to determine disease-associated changes in the immune system.ResultsThere are no differences between autologous aMSCs from IPF patients and HC in their stem cell properties, self-renewal capacity, osteogenic differentiation, secretome content, cell cycle inhibitor marker levels and mitochondrial health. IPF patients had altered peripheral blood immunophenotype including reduced B cells subsets, increased T cell subsets and increased granulocytes demonstrating disease-associated alterations in the immune system.ConclusionsOur results indicate that there are no differences in aMSC properties from IPF patients and HC, suggesting that autologous aMSCs may be an acceptable option for IPF therapy. The altered immune system of IPF patients may be a valuable biomarker for disease burden and monitoring therapeutic response,

Potential Disease-Modifying Activity of Navtemadlin (KRT-232), a First-in-Class MDM2 Inhibitor, Correlates with Clinical Benefits in Relapsed/Refractory Myelofibrosis (MF)

Potential Disease-Modifying Activity of Navtemadlin (KRT-232), a First-in-Class MDM2 Inhibitor, Correlates with Clinical Benefits in Relapsed/Refractory Myelofibrosis (MF)

Abstract B02: 5-Hydroxymethylcytosine profiles in circulating cell-free DNA are biomarkers of disease burden in children with neuroblastoma

Abstract Background: 5-Hydroxymethylcytosine (5-hmC) is an epigenetic marker of open chromatin and active gene expression. The level of 5-hmC expression serves as a biomarker in adults with cancer, and we recently demonstrated the prognostic significance of tumor whole-genome 5-hmC profiling in children with neuroblastoma. Here, we used Nano-hmC-Seal technology to profile 5-hmC in circulating cell-free DNA (cfDNA) in blood samples from patients with neuroblastoma and age-matched healthy children. Methods: Plasma was collected from 32 children with neuroblastoma at various time points during therapy and 34 well children. 5-hmC sequencing was performed using 100ng of cfDNA extracted from plasma. Metastatic disease burden was classified as high, moderate, low, or none based on Curie MIBG scores, the percentage of tumor cells in bone marrow samples, and/or measurable disseminated soft tissue disease. Results: One hundred cfDNA 5-hmC profiles were generated from 27 high-risk, 3 intermediate-risk, and 2 low-risk patients and 34 well controls. Hierarchical clustering based on 5-hmC levels of 347 genes separated the samples into three main clusters corresponding to high, moderate, and low/no disease burden in the bone, bone marrow, and/or soft tissue. Using functional gene pathways, two subsets of patients within the low/no cluster were revealed that correlated with low tumor burden or no clinically detectable metastatic disease (sensitivity 70%, specificity 76.9%). 5-hmC profiles tracked closely with changes in disease status in 15 patients with serial samples. Samples from patients with a detectable tumor burden had genes with elevated 5-hmC in pathways of neuronal stem cell maintenance and the epigenetic regulatory complexes PRC2 and CTCF/cohesion. Conclusions: cfDNA 5-hmC profiles correlate with disease burden in children with neuroblastoma and may serve as sensitive biomarkers of treatment response. Analysis of transcriptional networks regulated by these epigenomic modifications may lead to a deeper understanding of the pathways that drive resistance to treatment. Citation Format: Mark A. Applebaum, Erin K. Barr, Jason Karpus, Meritxell Oliva, Alexandre Chlenski, Helen R. Salwen, Emma Wilkinson, Marija Dobratic, Elizabeth A. Sokol, Barbara E. Stranger, Chuan He, Susan L. Cohn. 5-Hydroxymethylcytosine profiles in circulating cell-free DNA are biomarkers of disease burden in children with neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B02.

Cerebral atrophy in amyotrophic lateral sclerosis parallels the pathological distribution of TDP43.

Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by a preferential involvement of both upper and lower motor neurons. Evidence from neuroimaging and post-mortem studies confirms additional involvement of brain regions extending beyond the motor cortex. The aim of this study was to assess the extent of cerebral disease in amyotrophic lateral sclerosis cross-sectionally and longitudinally and to compare the findings with a recently proposed disease-staging model of amyotrophic lateral sclerosis pathology. Deformation-based morphometry was used to identify the patterns of brain atrophy associated with amyotrophic lateral sclerosis and to assess their relationship with clinical symptoms. Longitudinal T1-weighted MRI data and clinical measures were acquired at baseline, 4 months and 8 months, from 66 patients and 43 age-matched controls who participated in the Canadian Amyotrophic Lateral Sclerosis Neuroimaging Consortium study. Whole brain voxel-wise mixed-effects modelling analysis showed extensive atrophy patterns differentiating patients from the normal controls. Cerebral atrophy was present in the motor cortex and corticospinal tract, involving both grey matter and white matter, and to a lesser extent in non-motor regions. More specifically, the results showed significant bilateral atrophy in the motor cortex and corticospinal tract (including the internal capsule and brainstem) and ventricular enlargement, along with significant longitudinal atrophy in precentral gyrus, frontal and parietal white matter, accompanied by ventricular and sulcal enlargement. Atrophy in the precentral gyrus was significantly associated with greater disability as quantified with the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (P < 0.0001). The pattern of atrophy observed using deformation-based morphometry was consistent with the Brettschneider’s four-stage pathological model of the disease. Deformation-based morphometry provides a sensitive indicator of atrophy in Amyotrophic lateral sclerosis and has potential as a biomarker of disease burden, in both grey matter and white matter.

Glucosylsphingosine as a biomarker of disease burden in Gaucher disease

Poor medication adherence is associated with adverse health outcomes and higher costs of care. However, inconsistencies in the assessment of adherence are found in the literature.To evaluate the effect of different measures of adherence in the comparative effectiveness of complex interventions to enhance patients' adherence to prescribed medications.A systematic review with network meta-analysis was performed. Electronic searches for relevant pairwise meta-analysis including trials of interventions that aimed to improve medication adherence were performed in PubMed. Data extraction was conducted with eligible trials evaluating short-period adherence follow-up (until 3 months) using any measure of adherence: self-report, pill count, or MEMS (medication event monitoring system). To standardize the results obtained with these different measures, an overall composite measure and an objective composite measure were also calculated. Network meta-analyses for each measure of adherence were built. Rank order and surface under the cumulative ranking curve analyses (SUCRA) were performed.Ninety-one trials were included in the network meta-analyses. The five network meta-analyses demonstrated robustness and reliability. Results obtained for all measures of adherence were similar across them and to both composite measures. For both composite measures, interventions comprising economic + technical components were the best option (90% of probability in SUCRA analysis) with statistical superiority against almost all other interventions and against standard care (odds ratio with 95% credibility interval ranging from 0.09 to 0.25 [0.02, 0.98]).The use of network meta-analysis was reliable to compare different measures of adherence of complex interventions in short-periods follow-up. Analyses with longer follow-up periods are needed to confirm these results. Different measures of adherence produced similar results. The use of composite measures revealed reliable alternatives to establish a broader and more detailed picture of adherence.

Retinal pathology occurs in stiff-person syndrome.

To evaluate whether structural and functional changes occur in the afferent visual system of patients with stiff-person syndrome (SPS) and whether these changes correlate with disease burden, given the high concentration of γ-aminobutyric acid receptors, which are generally thought to be involved in SPS pathogenesis, in the retina. In this single-center, cross-sectional study, patients with SPS and healthy controls (HCs) underwent optical coherence tomography (OCT), with a subset undergoing high- and low-contrast visual acuity (VA) assessments. Burden of disease was assessed via the number of body regions affected. Individuals with uncontrolled hypertension or comorbid neurologic or ophthalmologic disorders were excluded. Statistical analyses were performed using mixed-effects linear regression models. Thirty-five patients with SPS and 40 age- and sex-matched HCs underwent OCT. A subset of 23 patients with SPS and 28 HCs underwent VA assessments. Relative to HCs, patients with SPS had lower ganglion cell + inner plexiform layer (GCIPL) thicknesses (SPS: 74.36 µm [SD 5.7]; HCs: 76.33 µm [SD 4.2]; p = 0.005), inner nuclear layer thicknesses (SPS: 44.37 µm [SD 2.7]; HCs: 45.18 µm [SD 2.2]; p = 0.042), and 100% (SPS: 53 [SD 9.6]; HCs: 57.5 [SD 6.1]; p = 0.005), 2.5% (SPS: 24.35 [SD 10.1]; HCs: 30.16 [SD 7.7]; p = 0.006), and 1.25% contrast (SPS: 16.41 [SD 10.6]; HCs: 20.84 [SD 8.6]; p = 0.034) letter acuity scores. GCIPL thicknesses correlated with the number of body regions affected in SPS (decrease of 1.25 µm [95% confidence interval, -2.2 to -0.3 µm; p = 0.008] per additional body region affected). Retinal neuronal pathology can occur in SPS. OCT may have utility as a biomarker of disease burden in SPS.

Response to Anti-Bcma CAR T Cell Therapy Correlates with T Cell Exhaustion and Activation Status in T Cells at Baseline in Myeloma

Response to Anti-Bcma CAR T Cell Therapy Correlates with T Cell Exhaustion and Activation Status in T Cells at Baseline in Myeloma

Molecular Quantification of Tissue Mast Cell Burden in Systemic Mastocytosis: A New Approach for Diagnostics and Prognostication

Background: The somatic KIT D816V mutation leads to an activation of the receptor tyrosine kinase KIT and represents a diagnostic criterion for systemic mastocytosis (SM). In the majority of patients, only a very few KIT D816V+ mast cells (MC) or MC precursors, if any, are found in peripheral blood (PB) and bone marrow (BM) aspirates. In contrast, the MC count in BM biopsies is typically much higher. Melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) is widely used for detecting KIT mutations in biopsies.1 We have previously proposed dPCR as a new standard method of KIT D816V testing in SM that can also reliably quantify the variant allele fraction (VAF) in formalin-fixed paraffin-embedded (FFPE) material.2 While multilineage involvement of KIT D816V indicated by a high VAF in PB was associated with an aggressive clinical course,3-6 the mutant allele burden in tissue sections has not been studied in a large series of SM patients.Methods: The VAF of KIT D816V in the tissue was assessed by dPCR (PrimePCR ddPCR, Biorad) on DNA isolated from 211 FFPE BM sections from 116 patients with SM (median age 53 years; 58 females, 58 males) and 57 controls (lymphoma patients undergoing staging biopsy). A total of 91 SM patients were diagnosed with indolent SM (ISM) and 25 with advanced SM (aggressive SM, SM with associated hematologic neoplasm, and mast cell leukemia according to WHO criteria). Results of diagnostic samples were compared to qualitative analysis of KIT D816V clamp-PCR. BM MC infiltration was quantified in a representative area of the tryptase stained section and expressed as % of nucleated cells. Total tryptase levels in serum were determined by a fluoroenzyme-immunoassay.Results: The KIT D816V mutation burden in FFPE BM sections of SM patients showed marked differences between patients ranging from 0.027% to 60% VAF (median: 1.9%). The VAF in BM sections was largely independent from that in liquid specimen (PB and BM aspirate). In particular, a number of ISM patients showed substantially higher VAF in FFPE tissue than in liquid specimen (Figure 1A). In line with this, a higher correlation of KIT D816V mutation burden to BM MC infiltration and serum tryptase levels was observed for FFPE tissue (Spearman's coefficient of correlation r = 0.68 and r = 0.68 respectively; Figure 1B-C) than for liquid specimen (r = 0.48 and r = 0.58 respectively). When analyzing subgroups of SM, FFPE BM sections of patients with advanced SM had a significantly higher KIT D816V allele burden (median 23.40%) compared to patients with ISM (median 1.65%; p<0.001 Figure 1D). Finally, we assessed the sensitivity and specificity of dPCR for KIT D816V analysis in FFPE BM section compared to clamp PCR. While a specificity of 100% was observed for both assays, dPCR showed a significantly higher sensitivity to detect the mutation in BM tissue of ISM patients compared to clamp PCR (97% vs. 89%; p<0.05).Conclusion: dPCR is a sensitive method to detect KIT D816V and reliably quantify the mutant allele burden in FFPE BM sections of SM patients. The mutational burden in the tissue represents a new molecular marker of the disease burden in SM since it reflects both the multilineage involvement of KIT D816V and the MC burden while the mutation burden in PB primarily reflects the KIT D816V multilineage involvement. Although the tissue mutational burden in patients with advanced SM is higher compared to ISM, the difference is not as substantial as described for PB. Thus, the prognostic value of KIT D816V allele burden measurement in SM needs to be redefined for BM sections. Potential future applications of KIT D816V quantification in FFPE BM sections include treatment response monitoring and definition of high disease burden in smoldering SM (SSM). We propose to include dPCR-based KIT D816V mutant allele burden measurement in BM tissue as a new biomarker for disease burden in SM in future clinical trials.

Unraveling the Prenylation-Cancer Paradox in Multiple Myeloma with Novel Geranylgeranyl Pyrophosphate Synthase (GGPPS) Inhibitors.

Post-translational prenylation of the small GTP-binding proteins (GTPases) is vital to a plethora of biological processes, including cellular proliferation. We have identified a new class of thienopyrimidine-based bisphosphonate (ThP-BP) inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS) that block protein prenylation in multiple myeloma (MM) cells leading to cellular apoptosis. These inhibitors are also effective in blocking the proliferation of other types of cancer cells. We confirmed intracellular target engagement, demonstrated the mechanism of action leading to apoptosis, and determined a direct correlation between apoptosis and intracellular inhibition of hGGPPS. Administration of a ThP-BP inhibitor to a MM mouse model confirmed in vivo downregulation of Rap1A geranylgeranylation and reduction of monoclonal immunoglobulins (M-protein, a biomarker of disease burden) in the serum. These results provide the first proof-of-principle that hGGPPS is a valuable therapeutic target in oncology and more specifically for the treatment of multiple myeloma.

Lipocalin 2 is a Yap‐beta‐catenin target and a biomarker of disease burden in a murine model of hepatoblastoma

Hepatoblastoma is the most common type of pediatric liver malignancy. Despite advances in therapy, metastatic disease is still associated with a poor prognosis. Aberrations in the Wnt/b‐catenin signaling pathway due to mutations in CTNNB1 are the most prevalent mutations findings in these tumors. Recently mutations in the nuclear translocation of the Yes‐Associated‐Protein Pathway (YAP) has also been reported in hepatoblastomas. In fact b‐catenin‐Yap co‐activation was observed in 80% of hepatoblastoma patients. Hydrodynamic tail vein injection of active upregulated‐beta‐catenin and active‐YAP plasmids in mice also led to hepatoblastoma tumors. Gene array analysis using mRNA isolated from these mice tumors from tumor‐bearing livers had identified lipocalin 2 as one of the upregulated genes, with increased binding sites to both TCF4 and TEAD, which are transcription factors for beta‐catenin and YAP respectively. Lipocalin 2 is a small molecule protein being increasingly studied in the field of cancer. Previously we had shown that indeed human hepatoblastoma tumors showed notable lipocalin‐2 (LCN2) expression by immunohistochemistry. To address if the increased LCN2 expression observed in humans correlated with the mouse model of hepatoblastoma, liver lysates from wild type livers and mouse hepatoblastoma tumors were analyzed. The tumor‐bearing livers were found to have a 40‐fold increased lipocalin 2 expression by real time PCR. We next used ELISA to quantify the amount of serum lipocalin 2 in these animals. We saw increased values of Lipocalin 2 serum levels in mice with tumors compared to the wild type. In fact, in fact we found that serum lipocalin 2 levels was an indicator of the tumor burden in these mice. The liver weight to body weight ratio of the mice had a direct positive correlation with serum lipocalin 2. To address that lipocalin 2 was regulated by Wnt and Yap signaling, we preformed western blot analysis on human hepatoblastoma HepG2 cells, that had been transfected with beta‐catenin or Yap silencing RNA. Protein levels of lipocalin 2 were decreased in following beta‐catenin knockdown of both, although it was more pronounced upon beta‐catenin suppression of cells. Additionally, western blot preformed on liver lysates from hepatocyte specific beta‐catenin knock out mice, also showed decreased levels of lipocalin 2. In conclusion, we have shown lipocalin 2 to be regulated by beta‐catenin and Yap signaling in hepatoblastoma and may be useful as a biomarker of the disease burden in a hepatoblastoma mouse model. There is an implication that this may be a potential serum marker in human patients as well. The fluctuations of lipocalin 2 levels in the serum may be a direct result of alterations in beta‐catenin signaling and could be participating to oncogenesis.

18FDG-PET Positivity Post Radiotherapy is a Biomarker for Relapse in Patients With Plasmacytoma

Solitary plasmacytomas are characterised by focal proliferation of monoclonal plasma cells in either soft tissue or bone. The management of solitary plasmacytomas is distinct from other plasma cell dyscrasias with radiotherapy forming the mainstay of treatment, with curative intent. Precise diagnosis of a solitary plasmacytoma is therefore essential, to ensure that the patient is treated on the appropriate pathway. Patients with solitary plasmacytoma can also progress with multiple plasmacytomas or Multiple Myeloma. Established risk factors for progression from solitary plasmacytoma to myeloma/multiple plasmacytomas are raised light chain ratio, aberrant plasma cells in marrow and size of plasmacytoma. Imaging biomarkers of relapse remains an active area of investigation. 18F FDG PET-CT has emerged as a useful tool to diagnose plasmacytomas and stage patients, but data is limited on use of 18F FDG PET-CT to measure treatment response. In Plasmacytoma patients, the role of residual PET positivity post therapy as a biomarker for disease burden and correlation with biochemical results, is not yet known. Likewise, the role of 18F FDG PET-CT as a biomarker to estimate the cure fraction and as a marker of disease progression requires further study. To answer these questions we conducted a retrospective analysis of twenty-four patients, referred with a histological diagnosis of plasmacytoma, over a three year period. We recorded patient baseline characteristics, size and location of their plasmacytoma, imaging findings, paraprotein/light chain and bone marrow results. We then compared their 18F FDGPET-CT and skeletal surveys to analyse what proportion of patients were subsequently upgraded to myeloma on imaging alone and whether their treatment pathway had changed. We also recorded the proportion of patients with solitary plasmacytoma who progressed with progression recorded on 18F FDG PET CT scan on follow-up imaging or until the first sign of biochemical relapse. Progression free and overall survivals were then calculated from time of diagnosis of plasmacytoma. Of the 24 patients, 9 patients were imaged with both skeletal survey and FDG PET-CT. On comparing the imaging findings, FDG PET-CT detected additional lesions in 55.6% of cases (5/9 patients), which were missed by conventional radiography and significantly changed treatment pathway. Follow up post treatment PET/CT was performed in 15/24 (62.5%) of patients. During the follow up period we found that 50% (12/24) patients with plasmacytoma progressed to Myeloma. In 66.7% of these cases, (8/12 patients) post therapy FDG PET-CT was positive, and only 1 PET-CT negative patient progressed. Only 50% (6/12 patients) of patients had biochemical evidence of either persistent disease or relapse at the time of clinical progression suggesting that FDG PET-CT is a useful imaging tool to identify plasmacytoma patients at risk of progression to Myeloma.

P047 - Comparison of tissue versus plasma Vascular Endothelial Growth Factor as biomarker of disease Burden in prostate cancer

P047 - Comparison of tissue versus plasma Vascular Endothelial Growth Factor as biomarker of disease Burden in prostate cancer

Abstract 3133A: Circulating tumor DNA as “liquid biopsy” in pediatric osteosarcoma

Abstract BACKGROUND: Osteosarcoma (OS) is the most common primary malignant bone tumor and children and adolescents represent half of new cases diagnosed each year. During and after treatment there is no non-invasive test to assess disease response and early relapse, respectively. Monitoring of circulating tumor material as a “liquid biopsy” has potential to provide this critical information in OS, as it has proven useful in other solid tumors. We hypothesized that circulating tumor DNA (ctDNA) can be extracted from mouse plasma and identified using next generation sequencing. This could ultimately be used to assess tumor burden, evaluate response to treatment, and monitor for recurrence in OS. We overcome the challenge of identifying tumor DNA in a background of host DNA by first using primary tumor material to identify genomic aberrations in a targeted gene region known to be altered in &amp;gt;95% of patients with OS. We then use next generation sequencing to identify in circulation the rare tumor DNA based on known aberrations expected to be present when there is a tumor burden. MATERIALS AND METHODS: Human osteosarcoma cell lines 143b and M17 were grown in culture and introduced into SCID mice via tail vein, subcutaneous flank, and tibial plateau injections. Terminal bleeds were performed 30 minutes after tail vein injection and at max tumor growth respectively. DNA was extracted from primary tumor cell lines as well as from the plasma of injected mice. We then performed next generation sequencing (NGS) with the Illumina HiSeq 2000 using custom designed probes capturing genes commonly altered in osteosarcoma including TP53, RB1, ATRX, DLG2, MET, PTEN, and SLC19A1. DNA extracted from the plasma of a SCID mouse that did not receive tumor cell injection was used as a negative control. RESULTS: Gene coverage of approximately 80% was obtained for targeted genes to a depth ranging from 250x to 2000x coverage for tumor cell lines. Cell free circulating tumor DNA was identified in plasma of mice injected with 143b mice via tail vein and flank injections but not from tumor cell free plasma negative control. Over 1000 mutations were identified, most notably c.467G&amp;gt;C p.R156P, a well documented osteosarcoma mutation, which was present in the 143b cell line as well as the DNA extracted from the plasma of tail vein and flank injected mice. CONCLUSIONS: Circulating cell free tumor DNA can be successfully extracted from SCID mouse plasma and identified using next generation sequencing of target genes. Based on these mouse findings, we anticipate that this will serve as a non-invasive biomarker of disease burden and response to therapy, as well as a biomarker to assess recurrence. The pilot work provides rationale to expand these findings into clinical trials that can prospectively validate our methods and findings. Our approach has the potential to improve outcomes for a childhood cancer frequently associated with poor survival. Citation Format: Michael Fremed, Sajida Piperdi, Wendong Zhang, Shahina Maqbool, Brent Calder, Raquel Castellanos, Jonathan Gill, Michael Roth, Bang Hoang, David Geller, Richard Gorlick, Daniel Weiser. Circulating tumor DNA as “liquid biopsy” in pediatric osteosarcoma. [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 3133A.

Abstract B37: Detection of sarcoma circulating tumor cells using a size based microfiltration device

Abstract The inability to effectively treat hematogenous metastasis remains the largest challenge of sarcoma therapy. The ability to isolate, quantify, and study circulating tumor cells (CTC) in sarcomas has the potential to enhance our understanding of the biology of metastasis, altering the way we treat patients with high grade sarcomas. Using a size based filtration device, CellSieve™, we were able to successfully identify and quantify CTC in a metastatic xenograft model as well as in blood samples from patients with high grade sarcomas. In mice, we utilized our orthotopic implantation/amputation model of metastasis. Using a TdTomato-expressing Ewing sarcoma cell line and a cocktail of fluorescently labeled antibodies to vimentin and CD45, we were able to detect both CTC and circulating tumor clusters (CTCL) in mice with growing tumors and with metastatic disease, but not in mice immediately post-amputation. These experiments were augmented with samples collected from patients with high grade sarcomas. In 18 samples from 15 patients, we were able to identify both CTC and CTCL. Furthermore, we were also able to validate the presence of CTC by PCR amplification of sarcoma-associated chromosomal translocations. Future work will use this system to explore the biology of sarcoma metastasis by studying genetic, transcriptional, and epigenetic characteristics of CTC and CTCL and comparing these cells to those in the bulk primary tumor. We will also further explore the possibility that CTC and/or CTCL might serve as a biomarker of disease burden, response to treatment, or metastatic risk. Citation Format: Masanori Hayashi, Peixuan Zhu, Catherine M. Albert, Diana Steppan, Gregory McCarty, Kyle W. Jackson, Cha-Mei Tang, David M. Loeb. Detection of sarcoma circulating tumor cells using a size based microfiltration device. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B37.

Pre-treatment circulating tumor DNA as a biomarker for disease burden in diffuse large B cell lymphoma (DLBCL).

8539 Background: Patients with DLBCL reveal striking heterogeneity in clinical outcomes. Plasma cell-free DNA (cfDNA) levels have been shown to be adversely prognostic. Both circulating tumor DNA (...

Circulating YKL‐40 in myelofibrosis a potential novel biomarker of disease activity and the inflammatory state

Chronic myeloproliferative neoplasms (MPN), encompassing essential thrombocythaemia (ET), polycythaemia vera (PV) and myelofibrosis (PMF), are featured by a chronic inflammatory state which is pronounced in myelofibrosis The value of YKL-40 as a biomarker of disease burden has been demonstrated in several different diseases, including cancer, diabetes mellitus and cardiovascular diseases. A state of chronic inflammation is shared by them all, YKL-40 also being involved in the severity of chronic endothelial inflammation, which today is considered of crucial importance for the development of atherosclerosis. The MPNs being cancers with a heavy burden of cardiovascular diseases we hypothesised that circulating YKL-40 might reflect the inflammatory process and potentially serve as a novel disease marker. Using ELISA, we measured YKL-40 in 15 patients with ET, 16 patients with PV, 17 patients with PMF and 30 healthy controls. YKL-40 was significantly elevated in PMF vs. control subjects, PMF levels median 43 ng/mL vs. controls median 28 ng/mL, P = 0.033. An increase from ET over PV may reflect the integrated impact of disease processes in MPNs.

Circulating tumor cells as a possible marker for micrometastatic disease in patients with localized pancreatic cancer.

175 Background: Circulating tumor cells (CTCs) are cells shed into circulation from tumors. They are increasingly recognized to be important biomarkers of disease burden in patients with solid tumors. Studies in pancreatic ductal adenocarcinoma (PDAC) have been limited due in part to low sensitivity of existing assays with extremely low numbers detected (0-15 per 7.5-15ml of blood). The development of newer microfluidic platforms has resulted in the ability to detect substantially greater numbers of CTCs. Methods: 15 patients with PDAC were enrolled in an IRB-approved study. Blood was collected in EDTA tubes and processed within 3 hours. CTCs were selected from 2-3ml of whole blood using monoclonal antibodies against epithelial cell adhesion molecule (EpCAM) and enumerated using a novel microfluidic platform. CTCs were confirmed by DAPI, CK8/18/19 and CD45 staining. Results: Patients with metastatic disease (n=12) had a mean of 29.7, median of 22 (3.5-107) CTCs per 1ml of blood. Patients with localized disease (n=3) had a mean of 3.8, median of 2.9 (2.3-6.2) CTCs per 1ml of blood. 0.5-1 CTCs per 1ml were detected in normal controls (n=2). The number of CTCs was significantly different between localized, metastatic and normal patients (p=0.01). 1 patient, initially thought to have localized disease by standard imaging but found to be metastatic at time of operation, had a mean of 45.9 CTCs per 1ml of blood compared to a mean of 3.8 CTCs per 1ml in patients who underwent a curative resection (p=0.009). Conclusions: Studies of CTCs in PDAC have been very limited. Our ability to detect large numbers of CTCs with good dynamic range suggests that further investigation into CTCs as a prognostic marker in PDAC is warranted. This is the first study that we are aware of to find CTCs in patients with localized disease. The presence of CTCs in patients with localized PDAC is surprising and may be associated with findings of unexpected metastatic disease at surgery. Further follow-up will be needed to determine if the presence of CTCs in these patients is a harbinger of shorter progression-free survival and overall survival after curative operations.