The Analysis of Cell-Free DNA Concentrations and Integrity in Serum of Initial and Treated of Lymphoma Patients T

Clinical Biochemistry 63 (2019) 59–65 Contents lists available at ScienceDirect Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem The analysis of cell-free DNA concentrations and integrity in serum of initial and treated of lymphoma patients T Jianqiu Wua, Weiyan Tanga, Laiquan Huangb, Ning Houc, Jing Wud, Xianfeng Chenge, Dawei Mac, ⁎ Pudong Qianf, Qian Sheng, Wenjie Guof, Wei Penga, Yufei Liua, Cunshun Jiangh, Jifeng Fenga, a Department of Medical Oncology, Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research & The Affiliated Cancer Hospital of Nanjing Medical University, No.42, Baiziting, Nanjing City 210009, Jiangsu Province, China b Department of Hematology, The First Affiliated Hospital of Wannan Medical College, No.2, Zheshan West Road, Wuhu City 241001, Anhui Province, China c Department of Pathology, Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research & The Affiliated Cancer Hospital of Nanjing Medical University, No.42, Baiziting, Nanjing City 210009, Jiangsu Province, China d Soochow University, No.1, Shizi Street, Suzhou city 215006, Jiangsu Province, China e Clinic laboratory of Institute of Dermatology and Hospital for Skin Diseases, Chinese Academy of Medical Sciences, No.12, Jiangwangmiao Street, Xuanwu District, Nanjing City 210042, Jiangsu Province, China f Department of Radiotherapy, Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research & The Affiliated Cancer Hospital of Nanjing Medical University, No.42, Baiziting, Nanjing City 210009, Jiangsu Province, China g Department of Medical Oncology, Nantong Tumor Hospital, No.48, Qingnian West Road, Nantong city 226000, Jiangsu Province, China h Department of Medical Oncology, Lanxi County People's Hospital. No.65, North Street of Langxi County, 242100, Anhui Province, China ARTICLE INFO ABSTRACT Keywords: Objective: To evaluate cell-free DNA (cfDNA) in plasma as a promising biomarker for lymphoma, altered levels of Lymphoma cfDNA and its association with clinical parameters are investigated in patients suffered from lymphomas. Cell-free DNA Methods: Peripheral blood specimens were collected from 60 patients with lymphoma during initial diagnosis ROC curve and those of another 107 patients with lymphoma during treated stage were also collected, 93 healthy volunteers Quantitative PCR were selected as control group. Quantitative PCR was used to detect cfDNA level in each group, cfDNA level in different groups was analyzed to understand its relationship with lymphoma patients' clinical features. After correlation analysis between cfDNA and clinical characteristics, Receiver operator characteristic curve was performed to analyze sensitivity and specificity of cfDNA and LDH. Results: cfDNA concentration and integrity in initial stage of lymphoma patients were significantly higher than those in treated stage, and cfDNA concentration in treated phase was significantly higher than cfDNA concentration in control group. There was no significant difference in cfDNA integrity at treated stage compared with control group. There was no significant correlation between patient's age, gender, extranodal invasion and lymphoma pathological type and cfDNA concentration and integrity; In contrast, there was a significant correlation between ECOG score, LDH content, Ann Arbor stage, IPI, B-symptoms, Ki-67 expression and radiotherapy and cfDNA concentration and integrity, both at the time of initial diagnosis and treated stage. cfDNA concentration detection is an optimal diagnostic indicator, followed by cfDNA integrity detection, the sensitivity and specificity of both are superior to the traditional LDH detection. Conclusion: cfDNA level is significantly increased in lymphomas patient plasma and may help lymphoma screening. cfDNA level may serve as a potential indicator of lymphomas treatment efficacy. 1. Introduction cases of malignant lymphoma in Europe and the United States have been increasing year by year, and the annual incidence rate is as high as Lymphomas is malignant tumors that originate in lymph nodes and 11/100000–18/100000. In China, lymphoma accounts for about 4% of extranodal lymphoid tissues. According to the different histopatholo- new malignant tumors each year; lymphoma accounts for about half of gical features, they can be divided into two categories: Hodgkin's the newly diagnosed blood system tumors each year. The morbidity of lymphoma and non-Hodgkin's lymphoma [1]. In recent years, new lymphoma is only about 6.4/100000, but it has the high malignancy ⁎ Corresponding author. E-mail address: [email protected] (J. Feng). https://doi.org/10.1016/j.clinbiochem.2018.10.002 Received 29 March 2018; Received in revised form 13 September 2018; Accepted 3 October 2018 Available online 04 October 2018 0009-9120/ © 2018 Published by Elsevier Inc. on behalf of The Canadian Society of Clinical Chemists. J. Wu et al. Clinical Biochemistry 63 (2019) 59–65 and poor prognosis [2]. The clinical manifestations of lymphoma are was transferred to a new tube and centrifuged at 16000g for 10 min. varied including painless mass or fever night sweats, vomiting, weight Purified plasma was carefully removed without disturbing lower re- loss and other systemic symptoms [3]. The risk factors of lymphoma are sidual layer. Minimum aliquot 200 μL plasma were used for DNA ex- known to be infection, immunodeficiency and so on, but the exact cause traction immediately or stored at −80 °C freezer. Plasma samples were is still unknown [4]. In the past decades, lymphoma was characterized thawed on ice and spun at 10000 g for 3 min before DNA purification. and classified into different subgroups according to the morphology. DNA was purified from 200 μL of plasma and eluted by 50 μL elution Recent discoveries in genetics and molecular biology have re- buffer using QIAamp DNA Blood Mini Kits (Qiagen, Valencia, CA) ac- volutionized our understanding of the initiation and progression of cording to the manufacturer's instructions. DNA samples were ready to lymphoma. Various new and potentially powerful molecular markers use for quantification or stored at −20 °C freezer. for lymphoma have been identified by researchers. However, there is a need for a universal biomarker available for all lymphoma patients of 2.3. Quantitative Polymerase Chain Reaction (QPCR) different subgroups. Cell-free DNA is a newly discovered biomarker in cancer research. It QPCR was performed on a LightCycler LC480 PCR machine (Roche refers to a nucleotide fragment in plasma that has a DNA double-helical Molecular Systems, Inc. Pleasanton, CA, USA). To measure the con- structure. In recent years, with the rapid progress of molecular genetics, centration of plasma cfDNA, the repetitive LINE 1(Long interspersed basic research and clinical research of cfDNA has been gradually dee- nuclear element 1) 97 bp (both for short and long) and LINE1 300 bp pened, including non-tumor-specific plasma DNA content and tumor- (only for long) DNA fragments were amplified as described respectively specific gene level abnormalities [5]. According to reports in the lit- by [7]. The LINE1 97 bp primer amplified apoptotic and non-apoptotic erature, cfDNA plays an important role in the diagnosis, treatment, and DNA fragments while the LINE1 300 bp primer amplified non- apop- prognosis of so many tumors, and it replaces tissue biopsy with a simple totic DNA fragments only. The total amount of plasma DNA was re- blood test [6]. The occurrence of this liquid biopsy overcomes the in- presented by the QPCR result with LINE1 97 bp primer. DNA integrity convenience in acquisition of tumor tissue, and there is little research index was calculated as the ratio of LINE1 300 and LINE1 97 QPCR on quantitative monitoring of cfDNA in lymphoma in China. To further result. A serial diluted standardized solution of human genomic DNA understand the value of cfDNA in lymphomas, we used real-time (Thermo Fisher Scientific, Waltham, MA, USA) was used as a standard quantitative PCR to detect the content of cfDNA and explored the im- curve reference. The concentration of cfDNA in each sample was cal- portant role of cfDNA in lymphoma screening. culated according to the standard curve. QPCR reaction was performed in triplicate and mean values across triplicates were used for further 2. Materials and methods analysis. The mixture of QPCR reaction was in 20 μL volume contained 1 μL DNA template, 0.5 μL of the each forward and reverse primer 2.1. Case selection (LINE1 97 or LINE1 300), 10 μL UltraSYBR Mixture (Cwbiotech, Beijing, China) and 8 μL double-distilled water. Cycling conditions were 60 patients which newly diagnosed by pathological examination 1 min at 95 °C, and 35 cycles of 95 °C for 8 s, and 60 °C for 15 s. Each after lymph node resection from June 2017 to March 2018 were se- plate consisted of a plasma DNA sample and a negative control (water lected (24cases of diffuse large B-cell lymphoma, 11 cases of Hodgkin's template) and 7 serial diluted standard DNA solutions. lymphoma, 9 cases of follicular lymphoma, 8 cases of NK-T cell lymphoma, 2 cases of mucosal-associated lymphoid tissue lymphoma, 3 2.4. Immunohistochemistry staining cases of mantle cell lymphoma and 3 cases of peripheral T cell lymphoma). Among lymphoma patients, 32 were males and 28 were fe- Immunohistochemistry (SP) method was used for Ki-67 detection. males, aged 12 to 80 years, with a median age of 46 years. At the same The PBS was used instead of the primary antibody as a negative control. time, another 107 lymphoma patients which experienced treated stage The antigen was repaired using microwave method and the kit in- during August 2017 to March 2018 were selected (45 cases of diffuse struction was strictly followed. The positive expression of Ki-67 was large B-cell lymphoma, 20 cases of Hodgkin's lymphoma, 14 cases of located in the nucleus and stained with clear brown-yellow particles. follicular lymphoma, 15 cases of NK-T cell lymphoma, and 4 cases of Positive staining was observed in 5 high-magnification (×400) fields mucosal-associated lymphoid tissue lymphoma, 4 cases of mantle cell with 100 cells in each field and a total of 500 tumor cells.

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