Dogs were recruited from patients presenting to the Texas A&M University Veterinary Medical Teaching Hospital (TAMU VMTH) for routine wellness exams or from dogs owned by TAMU VMTH personnel. All animal studies were approved by the Texas A&M University Animal Care and Use Committee (AUP #2017-0350). Owners were questioned to determine the health status of each patient. In order to be eligible for inclusion, dogs were required to be over one year of age and healthy. Dogs were excluded if there was any secondary significant inflammatory/infectious disease or history of neoplasia. Information recorded for each patient included signalment, body weight, body condition score, and any relevant comorbidities reported by the owner.
The LSA dog cohort was recruited in part from dogs presenting to the TAMU VMTH for treatment of naive multicentric LSA (AUP #2019-0211). The remaining samples were recruited from the National Cancer Institute Division of Cancer Treatment and Diagnosis (NCI-DCTD) Canine Tumor Repository. When available, information including patient signalment, body weight, body condition score, stage of disease, and immunophenotype were recorded.
Samples were collected longitudinally from three dogs undergoing treatment for multicentric LSA at the TAMU VMTH (AUP #2019-0211). These dogs were selected to evaluate the duration and pattern of elevated cancer associated nucleosome concentrations in plasma. Blood was collected at each clinic visit. These samples were collected prior to treatment and labeled according to the visit date. Additional information including treatment protocol and remission status at each visit were recorded.
Sample Collection and Processing
For patients presenting to the TAMU VMTH, blood was collected and immediately placed in K2-EDTA blood collection tubes. Within one hour of collection, samples were centrifuged at room temperature at 3000xg for 10 min. Plasma was then immediately removed without disrupting the buffy coat layer, placed in pre-labeled cryovials and frozen at -80°C to run in batches. Processing samples with this protocol was shown to be appropriate for reliable, consistent nucleosome detection in dog plasma . Samples received from the DCTD Canine Tumor Repository were stored frozen at -80°C to be run in batches.
Frozen samples were thawed and allowed to come to room temperature for at least 30 minutes prior to analysis. All samples were performed in duplicate. The samples were evaluated using the Nu.Q™ H3.1 ELISA (Belgian Volition, SRL, Isnes, Belgium) and were performed according to the manufacturer’s instructions. Briefly, a standard curve was generated using the known standards provided. Before use, the wells were washed 3 times with 200μL of the provided diluted wash solution with excess solution being removed after each wash. Patient and healthy dog plasma samples were vortexed and then centrifuged for 2 min at 11,000xg at 4°C before samples were loaded into the plates. Lymphoma samples were diluted 3-fold in order to ensure that they would register on the plates within the limits of the colorimetric standards. Twenty microliters of patient samples and kit controls were run in duplicate in wells on 96 well plates. Eighty microliters of assay buffer was then added to each well. The plates were sealed with foil and incubated at room temperature for 2.5 hours under agitation at ~700rpm. Plates were emptied and washed as described above. Next, 100μL of HRP labelled detection antibody was added to each well. The plate was sealed with foil and incubated at room temperature for 1.5 hours under agitation at ~700rpm. Plates were then emptied and washed as described above. Next, 100μL of TMB substrate was added to each well. The plate was sealed with foil and incubated at room temperature for 20 minutes in the dark under agitation at ~700rpm. One hundred microliters of stop solution were then added and the plate was shaken gently. Plates were read at an absorbance of 450nm (BioTek Synergy H1 plate reader, BioTek Instruments, Winooski, VT) within 5 minutes of stop solution being added. The standard curve was linearized and fitted to a 5-parameter logistic curve using statistical software (Graphpad Software, version 8, San Diego, CA).
For the longitudinal LSA patients, samples were submitted to the Texas A&M University Gastrointestinal Laboratory for their commercially available CRP assay if sufficient sample quantity was present. If sample quantity was not sufficient for both nucleosome and CRP analysis, nucleosome assays were given priority.
Thymidine Kinase Assays
The Canine Thymidine Kinase 1 soluble ELISA assay (My Biosource Inc, San Diego, CA) was used to evaluate TK levels in all dogs that were followed longitudinally. The assay was performed according to the manufacturer’s protocol. Briefly, 40μl of sample was added to wells followed by 10μl anti-TK1 antibody. Then 50μl streptavidin-HRP was added to each well except the blank control well. The plate was mixed well, covered with sealer and incubated for 60 minutes at 37°C. The plate was then washed 5 times with wash buffer and the wells were soaked with at least 0.35 ml wash buffer for 30 seconds to 1 minute for each wash. Next 50μl of substrate solution A was added to each well followed by 50μl of substrate solution B to each well. The plate was covered with a fresh sealer for 10 minutes at 37°C in the dark. Finally, 50μl of Stop Solution was added to each well. Plates were read at an absorbance of 450nm (BioTek Synergy H1 plate reader, BioTek Instruments, Winooski, VT) within 10 minutes of stop solution being added. The standard curve was linearized and fitted to a 5-parameter logistic curve using statistical software (Graphpad Software, version 8, San Diego, CA).
Descriptive statistics for the patient populations were performed using Microsoft excel for Mac (v. 16.16.27, 2016). For data sets containing only two cohorts, such as the healthy controls versus all LSA cases, a Wilcoxon rank sum test was used to compare the medians of the data sets. For data sets where multiple conditions were compared such as disease stage, a two-way ANOVA for repeat measures with a Tukey’s multiple comparisons test was performed. This part of the analysis was performed using GraphPad Prism version 8.0.0 for Macintosh, GraphPad Software, San Diego, California USA, www.graphpad.com. Spearman’s correlation, ROC curves and specificity/sensitivity calculations were performed using R version 3.4.3 and the pROC package [35, 36].