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Yanchun Qu,1– 3,* Yufeng Liu,3,* Kailin Ding,3 Yong Li,1,2 Xiaoyu Hong,4 Haibo Zhang1,2 1Department of Oncology, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 2Department of Oncology, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou, People’s Republic of China; 3The Second Clinical Medical College of Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 4Nanjing Geneseeq Technology Inc, Nanjing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Haibo ZhangDepartment of Oncology, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, No. 111 Dade Road, Yuexiu District, Guangzhou, 510120, People’s Republic of ChinaTel +86-020-81887233Email haibozh@gzucm.edu.cnAbstract: Human epidermal growth factor receptor2 (HER2) overexpression/amplification is associated with high malignancy, rapid disease progression and poor overall survival in breast cancer. The application of anti-HER2 drugs has greatly improved the survival of patients with HER2-positive breast cancer, but drug resistance issues affect the long-term efficacy. The HER2 mutation is considered to be one of the reasons for resistance to anti-HER2 therapy, and there is currently no standard treatment. We report for the first time the detection of HER2 amplification with R157W mutation by second-generation sequencing (NGS) in a 57-year-old hormone receptor-negative, HER2-positive woman with advanced breast cancer who was resistant to multi-line anti-HER2 therapies. She subsequently received pyrotinib combined with capecitabine treatment and achieved partial response. The small-molecule pan-HER family irreversible inhibitor pyrotinib combined with capecitabine has shown a promising effect in the treatment of HER2 mutation-induced resistance, but the molecular mechanism and efficacy need to be further verified.Keywords: breast cancer, HER2 amplification, HER2 R157W mutation, pyrotinib plus capecitabine, anti-HER2 treatment

Daqian Wan,1,2,* Yang Qu,3,* Lei Zhang,4,5 Songtao Ai,3 Liming Cheng1,2 1Department of Orthopedics, Tongji Hospital Affiliated to Tongji University School of Medicine, Shanghai, People’s Republic of China; 2Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration, Ministry of Education of the People’s Republic of China, Shanghai, People’s Republic of China; 3Department of Radiology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China; 4Department of Orthopedics, The First Affiliated Hospital of Shandong First Medical University, Shandong, People’s Republic of China; 5Department of Orthopedics, Shandong Provincial Qianfoshan Hospital, Shandong University, Shandong, People’s Republic of China*These authors contributed equally to this workCorrespondence: Songtao AiDepartment of Radiology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, No. 639 Zhizaoju Road, HuangpuDistrict, Shanghai, People’s Republic of ChinaTel +86 18019790801Email ai.songtao@qq.comLiming ChengTongji Hospital Affiliated to Tongji University School of Medicine, No. 389 Xincun Road, Putuo District, Shanghai, People’s Republic of ChinaTel +86 13701959550Email chlm.d@163.comIntroduction: Osteosarcoma is the most common primary malignant tumor in children and young patients. Although neoadjuvant chemotherapy and surgery could improve the prognosis of these patients, treatment outcomes are poor because of its low early diagnosis rate and high degree of malignancy as well as its tendency for early metastasis. In the field of osteosarcoma, lncRNAs have become a hot spot for studying the molecular mechanisms driving malignant biological characteristics and exploring effective treatment methods. An lncRNA is a long noncoding RNA lacking protein-encoding ability, and in its RNA form, it regulates various gene expression processes, such as epigenetic regulation, transcriptional regulation, and posttranscriptional regulation. LncRNAs play an important role in tumorigenesis and metastasis.Methods: We used bioinformatics software to analyze the data in geo database. CCK-8 and Transwell were used to detect the effect of lncRNA LINC00691 on the proliferation and migration of osteosarcoma cells. The target gene of LINC00691 was detected by bioinformatics analysis and RNA pull down.Results: In this study, we identified the lncRNA LINC00691 and confirmed its expression in osteosarcoma cells through GEO database analysis. Expression analysis showed that the levels of lncRNA LINC00691 in osteosarcoma cells were decreased compared to those of control cells. Overexpression of LINC00691 could inhibit the proliferation, migration, invasion, and induction of G1 cell cycle arrest in osteosarcoma cells, which was shown through in vitro and in vivo studies. Using bioinformatics analysis, RNA pull down experiments and luciferase reporter gene detection assays, we found that LINC00691 regulated ST5 expression by binding miR-1256. LINC00691 overexpression inhibited EMT by promoting the expression of E-cadherin and increasing the expression of ZEB1, Snail, and Fibronectin.Conclusion: These results suggested that overexpressed LINC00691 promoted the expression of ST5 by regulating the function of miR-1256 through a ceRNA mechanism. The LINC00691/miR-1256/ST5 pathway plays an important role in the progression and metastasis of osteosarcoma and represents a good therapeutic target.Keywords: osteosarcoma, ST5, lncRNA, miR-1256

Di Huang,1 Xue Chen,1 Xuan Chen,1 Yan Qu,1 Yuanyuan Wang,2 Yafei Yang,3 Yufeng Cheng1 1Department of Radiation Oncology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, 250012, People’s Republic of China; 2Department of Oncology, People’s Hospital of Linyi County, Dezhou, Shandong 251500, People’s Republic of China; 3Department of Hepatobiliary Surgery, Qilu Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, 250012, People’s Republic of ChinaCorrespondence: Yufeng Cheng Department of Radiation OncologyQilu Hospital, Cheeloo College of Medicine, Shandong University, 107 West Wenhua Road, Jinan, Shandong 250012, People’s Republic of China, Email qiluchengyufeng@126.comBackground: The WD40 protein family member WD repeat domain 5 (WDR5) plays significant roles in the tumorigenesis and development of multiple organ tumours. However, the correlation between WDR5 expression and oesophageal squamous cell carcinoma (ESCC) has not been elucidated.Methods: WDR5 mRNA expression data were acquired from The Cancer Genome Atlas (TCGA) database, and the expression and prognostic potential of WDR5 were assessed by immunohistochemistry (IHC) and Western blot. The cell counting kit-8 (CCK-8), colony formation assay and cell cycle evaluation were performed to verify the WDR5 function in vitro. The xenograft model was used to verify WDR5 function in vivo.Results: The mRNA and protein expression levels of WDR5 were significantly upregulated in ESCC tissues compared with expression in adjacent normal tissues. Kaplan–Meier analysis showed that high WDR5 expression in ESCC patients was associated with poor overall survival (P=0.004). Multivariate analysis revealed that WDR5 overexpression emerged as an independent predictor of poor overall survival (P=0.013) in ESCC. The in vitro and in vivo experiments revealed that downregulation of WDR5 expression blocked cell proliferation of ESCC. Mechanistically, we found that WDR5 may influence ESCC proliferation by targeting the PI3K/AKT/mTOR signalling pathway.Conclusion: Our data demonstrate that overexpression of WDR5 was associated with poor prognosis in patients with ESCC and that WDR5 may act as a potential novel prognostic biomarker for ESCC.Keywords: WDR5, oesophageal squamous cell carcinoma, prognosis, proliferation

Si Mi, Yan Qu, Xue Chen, Zhihua Wen, Pengxiang Chen, Yufeng Cheng Department of Radiation Oncology, Qilu Hospital of Shandong University, Jinan, Shandong, People’s Republic of ChinaCorrespondence: Yufeng Cheng Email shandong_sdu2019@126.comBackground: Dioxygenase 12-lipoxygenase (12-LOX) plays an important role in tumorigenesis and promotes angiogenesis and proliferation in several tumors, including prostate and breast tumors. Radiotherapy enhances the expression of 12-LOX in esophageal squamous cell carcinoma (ESCC). Two types of macrophages can be found in the tumor microenvironment. The M2 subtype accelerates tumor progression; however, the relationship between 12-LOX and macrophages is not well established. Here, we explore this interaction and its effect on ESCC to induce tumor progression.Methods and Results: RT-qPCR and Western blot analyses were used to evaluate the mRNA and protein expression levels of 12-LOX and chemokine (C-C motif) ligand 5 (CCL5) in ESCC after radiotherapy. CCL5 expression was increased by 12-LOX upregulation but was suppressed by the well-established 12-LOX inhibitor, baicalein. Furthermore, CCL5 attracted and repolarized human myeloid leukemia mononuclear cells (THP-1)-derived macrophages. Finally, ESCC co-culture with THP-1-derived macrophages led to a strong cancer migratory capacity.Conclusion: Radiation-induced 12-LOX overexpression in ESCC upregulates CCL5 expression, thereby attracting THP-1-derived macrophages and promoting their polarization to the M2 subtype, which enhances cellular metastasis.Keywords: radiotherapy, 12-LOX, CCL5, macrophage

Yunhui Qu,1 Jinbo Liu2 1Department of Clinical Laboratory, First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, People’s Republic of China; 2Department of Colorectal Surgery, First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, People’s Republic of ChinaCorrespondence: Jinbo Liu Email ljbzzuedu@163.comBackground: Increasing investigations indicate that long noncoding RNA (lncRNA) is responsible for diverse biological functions during the progression of cancer. However, its functions and underlying mechanisms remain elusive. Here, we investigated the MAFG-AS1- expression profile in esophageal squamous-cell carcinoma (ESCC) patients and explored its biological function and potential molecular mechanisms.Methods: qRT-PCR and the GEPIA data base were used to evaluate expression levels of MAFG-AS1 in ESCC tissue and cells. WST1-proliferation, -migration, and -invasion assays were performed to define the role of MAFG-AS1 in ESCC. Potential molecular mechanisms of MAFG-AS1 were investigated with online bioinformatic analysis, qRT-PCR, and rescue assays.Results: MAFG-AS1 was upregulated in 45 ESCC-tissue samples and cell lines compared to that of adjacent nontumor tissue and normal esophageal cells. Higher MAFG-AS1 expressionindicated poor survival. Gain- and loss-of-function experiments suggested that MAFG-AS1 promoted ESCC-cell proliferation, migration, and invasion. Molecular mechanism analysis and rescue assay showed that miR143 inhibitors partly abolished the suppression of MAFG-AS1 knockdown on EC109-cells proliferation. Moreover, we found that LASP1 specifically targeted miR143. Collectively, these data indicated that MAFG-AS1 served as a ceRNA to elevate LASP1 levels by sponging miR143, and played an oncogenic role in ESCC.Conclusion: Our research findings demonstrate that MAFG-AS1 is a key regulator through a novel MAFG-AS1–miR143–LASP1 axis in ESCC development and progression, which may offer a potential therapeutic target for ESCC.Keywords: lncRNA, ESCC, MAFG-AS1, miR143, LASP1

Xiuyan Yin,1,* Xiaohong Li,2,* Guijiao Feng,3,* Yuejie Qu,2 Hong Wang1 1Department of Ophthalmology, Yantai Yuhuangding Hospital, Yantai 264000, People’s Republic of China; 2Department of Obstetrics and Gynecology, Yantai Yuhuangding Hospital, Yantai 264000, People’s Republic of China; 3Department of the Outpatient, Yantai Yuhuangding Hospital, Yantai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yuejie Qu; Hong Wang Email cyg1816@163.com; 562205307@qq.comObjective: To detect LINC00565 expression level in endometrial carcinoma (EC) samples and cell lines, and the correlations between LINC00565 and clinical features of EC patients. After intervening LINC00565, the underlying mechanism about proliferative ability in EC cell lines is observed.Methods: Relative levels of LINC00565 and KLF9 in 52 paired EC and paracancerous tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between relative level of LINC00565 or KLF9 and clinical features of EC patients was analyzed. After knockdown of LINC00565 and KLF9, potential regulations of them on biological functions of EC were examined by Cell Counting Kit (CCK-8), colony formation assay and in vivo xenograft model in nude mice, respectively. At last, dual-luciferase reporter assay and rescue experiments were conducted to illustrate the mechanisms of LINC00565 and KLF9 in mediating the development of EC.Results: LINC00565 was upregulated in EC tissues. Chi-square analysis showed that a high level of LINC00565 predicted large tumor size, advanced pathological staging and poor prognosis in EC. Silence of LINC00565 decreased proliferative ability in EC cells and tumor growth in nude mice bearing EC. KLF9 was the target gene of LINC00565. The negative interaction between LINC00565 and KLF9 was responsible for stimulating the malignant development of EC. Knockdown of KLF9 could abolish the regulatory effects of silenced LINC00565 on proliferative ability and tumorigenesis in EC.Conclusion: LINC00565 is upregulated in EC tissues and closely linked to tumor size, pathological staging and poor prognosis in EC patients. LINC00565 stimulates proliferative ability in EC by downregulating KLF9.Keywords: LINC00565, KLF9, endometrial carcinoma, proliferation

Jiling Lv,1,2 Fang Zhang,3 Congying Zhai,2 Gejin Wang,4 Yan Qu1 1Department of Intensive Care Unit, Affiliated Qingdao Municipal Hospital of Qingdao University, Qingdao 266071, Shandong, People’s Republic of China; 2Department of Respiratory Medicine, The First Hospital of Zibo, Zibo 255200, Shandong, People’s Republic of China; 3Department of Radiotherapy, Yantai Affiliated Hospital of Binzhou Medical University, Yantai 26400, Shandong, People’s Republic of China; 4Department of Nursing, Zibo Vocational Institute, Zibo 255314, Shandong, People’s Republic of ChinaCorrespondence: Yan QuThe Intensive Care Unit, Qingdao Municipal Hospital Affiliated to Qingdao University, 1 Jiaozhou Road, Qingdao 266071, Shandong, People’s Republic of ChinaTel +8613668892506Email qdquyan@aliyun.comPurpose: B-cell lymphoma-2 (Bcl-2) associated athanogene 1 (Bag-1) is a multifunctional protein, and Bag -1 overexpression is associated with progression, metastasis, and drug resistance in lung cancer. This study assessed the effects of Bag-1 siRNA on sensitization of cisplatin on non-small cell lung cancer (NSCLC) cells.Material and methods: NSCLC A549 cell line was transfected with Bag-1 or negative control siRNA and then treated with cisplatin for cell viability, CCK-8, LDH, and flow cytometry assays. The Ca2+ levels were analyzed using Fluo-3/AM fluorescence staining, and the protein levels were assessed using Western blot analysis.Results: Bag-1 siRNA significantly knocked down Bag-1 expression and inhibited cell invasion versus the negative control siRNA, while Bag-1 silence sensitized cisplatin to induce A549 cells to apoptosis by induction of cell cycle G1 arrest. At protein level, Bag-1 silence reduced the expression ratio of Bcl-2 to Bcl-2 associated X protein (Bax), downregulated activity of the PI3K/AKT and mitogen-activated protein kinase (MAPK) pathways, and potently upregulated the calcium signaling-mediated pathway.Conclusion: This study demonstrated that Bag-1 silencing sensitized A549 to cisplatin to enhance A549 cell apoptosis by modified multiple gene pathways. Further study will evaluate the usefulness of Bag-1 siRNA as a potential targeting therapy for NSCLC.Keywords: bag-1, apoptosis, non-small cell lung cancer, chemotherapy

Yan Qu,1 Na Shao,2 Wenjing Yang,1 Jianbo Wang,1,* Yufeng Cheng1,* 1Department of Radiation Oncology, Qilu Hospital of Shandong University, Jinan, Shandong 250012, People’s Republic of China; 2Department of Oncology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, People’s Republic of China *These authors contributed equally to this work Objective: The main aim of this study was to investigate the association of polymorphisms in long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) with the risk of esophageal squamous cell carcinoma (ESCC) in a Chinese population.Methods: A total of 245 ESCC patients and 490 gender- and age-matched cancer-free controls were genotyped for four tag single nucleotide polymorphisms (SNPs) of MALAT1 (rs3200401 C>T, rs1122709 C>G, rs664589 C>G, and rs619586 A>G). Statistical analyses including chi-squared test and logistic regression were performed to identify the association between the tag SNPs and risk of ESCC, and false discovery rate (FDR) T polymorphism of MALAT1 was significantly associated with increased risk of ESCC (CT vs CC: adjusted OR =1.59, 95% CI =1.07–2.35, P=0.021; TT vs CC: adjusted OR =2.27, 95% CI =1.04–4.96, P=0.039; dominant model [CT+TT vs CC]: adjusted OR =1.68, 95% CI =1.16–2.43, P=0.006). In the stratified analysis, rs3200401 TT and CT/TT genotypes were associated with increased risk of ESCC compared with CC genotype in subgroup of never drinking (TT vs CC: adjusted OR =2.34, 95% CI =1.02–5.34, P=0.044; CT/TT vs CC: adjusted OR =1.52, 95% CI =1.02–2.26, P=0.041). However, compared with AA genotype, MALAT1 rs619586 GG was associated with decreased risk of ESCC in ever drinking subgroup (GG vs AA: adjusted OR =0.38, 95% CI =0.15–0.99, P=0.049). The results remained significant after FDR adjustment (FDR value T in MALAT1 gene is associated with increased risk of ESCC. Since the association between rs619586 A>G polymorphism and ESCC risk was not significant after FDR adjustment, there was a minor possibility that rs619586 A>G might be a protective factor for ESCC. Keywords: MALAT1, polymorphism, esophageal squamous cell carcinoma, single nucleotide polymorphism, susceptibility

Wenjing Yang, Yan Qu, Bingxu Tan, Yibin Jia, Nana Wang, Peng Hu*, Jianbo Wang* Department of Radiation, Qilu Hospital of Shandong University, Jinan, People’s Republic of China *These authors contributed equally to this work Purpose: IκB kinase epsilon (IKBKE; IKKε), a member of the nuclear factor-κB kinase inhibitor family, is upregulated in several human cancers, including breast cancer, prostate cancer, and ovarian cancer. Esophageal squamous cell carcinoma (ESCC) is one of the most common and most aggressively malignant cancers with dismal prognosis. However, the state of IKBKE expression in ESCC is still unknown and its potential value remains unexplored.Patients and methods: IKBKE protein expression was evaluated by immunohistochemistry in 118 paraffin specimens of ESCC treated by curative surgery. All patients were regularly followed up by telephone over 3 years after surgery. The chi-square test, Kaplan–Meier method, and Cox proportional hazard regression model were used to analyze the relationship of IKBKE expression, clinicopathological characteristics, and prognostic value for ESCC.Results: IKBKE expression was 61.9% (73/118) in paraffin-embedded archived ESCC. Its expression was significantly associated with tumor differentiation grade (p=0.045) and advanced TNM (pathologic tumor node metastasis) stages (p=0.023). In univariate analysis, IKBKE expression was closely associated with decreased 3-year disease-free survival (HR 1.804, 95% CI 1.076–3.027; p=0.023) and overall survival (HR 2.118, 95% CI 1.189–3.773; p=0.009). Meanwhile, in multivariate analysis it was identified as an independent prognostic factor for 3-year disease-free survival (HR 1.777, 95% CI 1.034–3.054; p=0.037) and overall survival (HR 2.078, 95% CI 1.138–3.796; p=0.017).Conclusion: Our data indicated for the first time that IKKε expression is a highly recurrent event in ESCC and could play a pivotal role in the evaluation of prognosis. IKBKE upregulation is negatively associated with disease-free survival and overall survival. Therefore, IKBKE could serve as a prognostic variable and potential therapeutic target for this malignancy. Keywords: IKBKE, esophageal squamous cell carcinoma, biomarker, prognosis, immunohistochemistry&nbsp

Pengxiang Chen,1 Cong Wang,1 Bo Cheng,2 Effat Un Nesa,1 Yuan Liu,1 Yibin Jia,1 Yan Qu,1 Ziying Jiang,1 Jie Han,3 Yufeng Cheng1 1Department of Radiation Oncology, Qilu Hospital of Shandong University, Jinan, 2Medical College of Qingdao University, Qingdao, 3Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong University, Jinan, People’s Republic of China Background: Evidence implies that preoperative plasma fibrinogen and serum albumin are associated with cancer prognosis. We aimed to explore the prognostic values of the score based on plasma fibrinogen and serum albumin levels (FA score) in non-small cell lung cancer (NSCLC), and to compare that with prognostic nutritional index (PNI).Patients and methods: In all, 182 patients pathologically diagnosed with NSCLC were included in this study. Kaplan–Meier survival analysis and multivariate analysis were used in the prognostic analyses.Results: High FA score was related to smoking (P=0.005), poor differential grade (P=0.002), and advanced T stage (P

Hongsheng Yu,* Yong Qu,* Qingjun Shang, Chao Yan, Peng Jiang, Xiang Wang, Donghai Liang, Tao Jiang Department of Oncology, Affiliated Hospital of Qingdao University, Qingdao, People’s Republic of China *These authors contributed equally to this work Objective: The objective of this study was to explore the clinical effects of low-dose splenic irradiation on locally advanced non-small-cell lung cancer (NSCLC) patients.Methods: Thirty-eight patients with stage III NSCLC were randomly divided into a control group and a combined treatment group. The control group only received chest three-dimensional conformal radiotherapy, while the combined treatment group received low-dose splenic irradiation followed by chest three-dimensional conformal radiotherapy after 6hours. T lymphocyte subsets of the blood cells were tested before, during, and after treatment once a week. The side effects induced by radiation were observed, and a follow-up was done to observe the survival statistics.Results: The ratio differences in CD4+ cells, CD8+ cells, and CD4+/CD8+ before and after treatment were not statistically significant (P>0.05) in both the groups. The immune indexes were also not statistically significant (P>0.05) before and after radiotherapy in the combined treatment group. However, the numbers of CD4+ cells and CD4+/CD8+ ratios before radiotherapy were higher than after radiotherapy in the control group. There were no differences in the incidence of radiation toxicities between the two groups; however, the incidence of grade III or IV radiation toxicities was lower, and the dose at which the radiation toxicities appeared was higher in the combined treatment group. The total response rate was 63.16% (12/19) in the combined treatment group vs 42.11% (8/19) in the control group. The median 2-year progression-free survival (15months in the combined treatment group vs 10months in the control group) was statistically significant (P0.05).Conclusion: Low-dose radiation can alleviate the radiation toxicities, improve the short-term efficacy of radiotherapy, and improve the survival of locally advanced NSCLC patients. Keywords: non-small-cell lung cancer, low-dose splenic irradiation, clinical effects, immune function, radiation toxicities&nbsp

Guixiu Jin,1,3 Jing Zhao,2 Hongyan Qi,2 Meng Lou,2 Xia Liu,2 Yu Qu,3 Lingjun Zhao,3 Weifeng Zhang,3 Jimin Shao,2 Huizhen Zhong31School of Medicine, Ningbo University, Ningbo, 2Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, Hangzhou, 3Department of Gynecological Oncology, Ningbo Women and Children's Hospital, Ningbo, People's Republic of ChinaBackground: The present study aims to investigate the subunit expression and enzyme activity of ribonucleotide reductase in cervical cancer patients, and detect the combined effect of the ribonucleotide reductase inhibitor gemcitabine and the chemotherapeutic agent carboplatin on cervical cancer cell lines.Methods: Using real-time polymerase chain reaction, Western blotting, and cytidine 5'-diphosphate reduction assays, we tested the expression and activity of ribonucleotide reductase in cervical cancer patients. The antitumor activity of gemcitabine and/or carboplatin treatments to Siha and Caski human cervical cancer cell lines were assessed by Cell Counting Kit-8 viability assay, EdU incorporation assay, immunofluorescence assay, flow cytometry assay, and Western blotting methods. Additionally, synergistic efficacy was quantitatively analyzed using a combination index based on the Chou-Talalay method.Results: The mRNA levels of three ribonucleotide reductase subunits were all upregulated in the cervical cancer tissues compared with normal tissues (P