Establishment of a Tumor-bearing Mouse Model Stably Expressing Human Tumor Antigens Survivin and MUC1 VNTRs

The eukaryotic vectors VR1012 expressing survivin or 33 tandem repeats of human mucin 1（MUC1）（VNTRs）,namely,VR1012-S and VR1012-VNTR（VNTR=variable number of tandem repeat）,were constructed by cloning survivin and VNTR genes into VR1012,respectively.The eukaryotic vector pEGFP expressing survivin and MUC1 VNTRs fusion gene pEGFP-MS was also constructed.Mouse melanoma cell line（B16） stably expressing survivin and MUC1 VNTRs（MS ＋ B16） was established by Lipofectamine-mediated transfection of pEGFP-MS into B16 cells.EGFP expression in MS ＋ B16 cells was observed using a fluorescent microscope and survivin and MUC1 VNTRs（MS） expression was confirmed by means of Western blot analysis.A syngenic graft tumor model was generated by subcutaneous injection of MS ＋ B16 cells into C57/BL6 mice and tumor size increased rapidly with time in a cell number dependent manner.After the third immunization,mice were challenged subcutaneously with 5×l0 5 MS ＋ B16 cells.Compared with that of the negative control immunized with phosphate-buffered saline（PBS）,a significant reduction of tumor growth was observed in groups immunized with survivin plasmid DNA and MUC1 VNTRs plasmid DNA.Thus,the suppression of subcutaneous tumor was antigen-specific.This model is useful for the development of tumor vaccines targeting survivin and MUCI VNTRs.

SIGNIFICANCE OF EXPRESS OF SOME NONHORMONAL ANTIGENS IN PANCREATIC ENDOCRINE TUMORS

Objective: To study the express of some nonhormonal antigens in pancreatic endocrine tumors. Methods: The nonhormonal antigens including Alpha subunit of human chorionic gonadotropin (α HCG), progesterone receptors (PR), 7B2, HISL 19, in normal pancreatic islets and in 52 cases of pancreatic endocrine tumors (PET) were investigated by immunohistochemistry. Results: It was found that HCG can be detected in PET but not in normal islet cells. HCG immunoreactivity was expressed by 3 of 28 (10.7%) benign PET and by 14 of 24 (58.3%) malignant PET.PR was found by 20 of 28(71.4%) benign PET and by 7 of 24 (29%) malignant PET. 7B2 was detected by 23 of 28 (82.1%) benign PET and by 13 of 24 (54.2%) malignant PET. HISL 19 was appeared by 23 of 28 benign PET and by 11 of 24 (46%) malignant PET. Golgitype persisted in 87.5% malignant tumors. Conclusion: The assay of nonhormonal antigens may be well defined the clinico pathological characteristics of PET.

New Concepts in Tumor Antigens:Their Significance in Future Immunotherapies for Tumors

The identification and molecular characterization of self-antigens expressed by human malignancies that are capable of elicitation of anti-tumor immune responses in patients have been an active field in tumor immunology. More than 2,000 tumor antigens have been identified, and most of these antigens are self-antigens. These significant progresses have led to the renaissance of tumor immunology and studies on anti-tumor immunotherapy. However, despite of the progress in the identification of self-tumor antigens, current antigen-specific immunotherapies for tumors are far less satisfied than expected, which reflects the urgent need to improve our understanding on self-tumor antigens. In order to develop more effective antigen specific anti-tumor immunotherapies and to monitor the responses to these immunotherapies in patients with tumors, many important fundamental questions need to be addressed. We propose for the first time that the studies in addressing the characteristics of self-tumor antigens and autoantigens are grouped as a new subject termed ＂antigenology＂. In this brief review, we would outline the progress in the identification of tumor antigens in solid tumors and hematologic malignancies, and overview the new concepts and principles of antigenology and their significance for future immunotherapies to these malignancies.

Tumor antigen and MHC expression in glioma cells for immunotherapeutic interventions

AIM: To investigate the expression of tumor-antigens and major histocompatibility complex(MHC)-machinery components in glioblastoma multiforme cell lines flow cytometry staining methods were applied.METHODS: Ten GBM cell lines(three commercially available: U-87 MG, U-138-MG and GMS-10 as well as seven newly established cell lines from individual patients in low-passages: HROG02, HROG04, HROG05, HROG06, HROG10, HROG13 and HROG17) were analyzed for expression of(Ⅰ) general and(Ⅱ) GBMrelated tumor antigens as well as of(Ⅲ) components of the MHC machinery by flow cytometry.RESULTS: All cell lines expressed MHC class?Ⅰ?with seven out of the ten being HLA-A02 positive. Four of the seven primary cell lines additionally expressedMHC class Ⅱ in a constitutive manner. Of note, after interferon gamma(IFN-γ) treatment, all seven cell lines expressed MHC class Ⅱ. The tumor associated antigens(TAA) EGFR and survivin were expressed at high levels in all cell lines; whereas MART-1, RHAMM, WT-1 and IL-13Rα were expressed by at least half of the cell lines and HER2/neu, MAGE-1 and tyrosinase were expressed only by few cell lines. However, all cell lines expressed at least two of the candidate antigens included into this analysis.CONCLUSION: No obvious differences between commercially available and newly-established cell lines were observed. Thus, the latter in low-passages are interesting for(therapy-) screening and immunotherapeutic strategies.

Comparisons of voided urine cytology, nuclear matrix protein-22 and bladder tumor associated antigen tests for bladder cancer of geriatric male patients in Taiwan, China

Aim： To compare the results of bladder tumor associated antigen （BTA TRAK）, nuclear matrix protein 22 （NMP 22） and voided urine cytology （VUC） in detecting bladder cancer. Methods： A total of 135 elderly male and 50 healthy volunteers enrolled in this study were classified into three groups： （i） 93 patients with bladder cancer; （ii） 42 patients with urinary benign conditions; and （iii） 50 healthy volunteers. BTA TRAK and NMP 22 kits were used to detect bladder cancer. Voided urine cytology was used to compare the sensitivity and specificity of the screening tests. Results： The sensitivity and specificity of cytology, BTA TRAK and NMP 22 were 24% and 97%, 51% and 73%, 78% and 73%, respectively. The level of NMP 22 increased with tumor grading. The BTA TRAK kit has the lowest sensitivity among the screening tests. The NMP 22 with the best sensitivity can be an adjunct to cytology for evaluating bladder cancer. Conclusion： The NMP 22 test has a better correlation with the grading of the bladder cancer than BTA TRAK. As cytology units are typically not available in hospitals or in outpatient clinics, NMP 22 might be a promising tool for screening bladder cancer.

Prognostic value of preoperative carcinoembryonic antigen/tumor size in rectal cancer

BACKGROUND Carcinoembryonic antigen(CEA)is a commonly used biomarker in colorectal cancer.However,controversy exists regarding the insufficient prognostic value of preoperative serum CEA alone in rectal cancer.Here,we combined preoperative serum CEA and the maximum tumor diameter to correct the CEA level,which may better reflect the malignancy of rectal cancer.AIM To assess the prognostic impact of preoperative CEA/tumor size in rectal cancer.METHODS We retrospectively reviewed 696 stage I to III rectal cancer patients who underwent curative tumor resection from 2007 to 2012.These patients were randomly divided into two cohorts for cross-validation:training cohort and validation cohort.The training cohort was used to generate an optimal cutoff point and the validation cohort was used to further validate the model.Maximally selected rank statistics were used to identify the optimum cutoff for CEA/tumor size.The Kaplan-Meier method and log-rank test were used to plot the survival curve and to compare the survival data.Univariate and multivariate Cox regression analyses were used to determine the prognostic value of CEA/tumor size.The primary and secondary outcomes were overall survival(OS)and disease-free survival(DFS),respectively.RESULTS In all,556 patients who satisfied both the inclusion and exclusion criteria were included and randomly divided into the training cohort(2/3 of 556,n=371)and the validation cohort(1/3 of 556,n=185).The cutoff was 2.429 ng/mL per cm.Comparison of the baseline data showed that high CEA/tumor size was correlated with older age,high TNM stage,the presence of perineural invasion,high CEA,and high carbohydrate antigen 19-9(CA 19-9).Kaplan-Meier curves showed a manifest reduction in 5-year OS(training cohort:56.7%vs 81.1%,P<0.001;validation cohort:58.8%vs 85.6%,P<0.001)and DFS(training cohort:52.5%vs 71.9%,P=0.02;validation cohort:50.3%vs 79.3%,P=0.002)in the high CEA/tumor size group compared with the low CEA/tumor size group.Univariate and multivariate analyses identified CEA/tumor size as an independent prognostic factor for OS(training cohort:hazard ratio(HR)=2.18,95%confidence interval(CI):1.28-3.73,P=0.004;validation cohort:HR=4.83,95%CI:2.21-10.52,P<0.001)as well as DFS(training cohort:HR=1.47,95%CI:0.93-2.33,P=0.096;validation cohort:HR=2.61,95%CI:1.38-4.95,P=0.003).CONCLUSION Preoperative CEA/tumor size is an independent prognostic factor for patients with stage I-III rectal cancer.Higher CEA/tumor size is associated with worse OS and DFS.

EXPRESSION OF SV40 Tag AND FORMATION Tag-p53 AND Tag-Rb COMPLEXES IN CHINESE BRAIN TUMORS

Objective: To investigate the expression of SV40 Tag and formation of Tag-p53 and Tag-Rb complexes in Chinese brain tumors. Methods: SV40 large tumor antigen (Tag) were investigated by immunoprecipitation, silver staining and Western blot in 65 cases of Chinese brain tumors and 8 cases of normal brain tissues. Tag-p53 and Tag-Rb complexes were screened by the same way in 20 and 15 Tag positive tumor tissues respectively. Results: Tag was found in all of 8 ependymomas and 2 choroid plexus papillomas, 90% (9/10) of pituitary adenomas, 73% (11/15) of astrocytomas, 70% (7/10) of meningiomas, 50% (4/8) of glioblastoma multiform, 33% (2/6) of medulloblastomas, 5 oligodendrogliomas, 1 pineocytoma and 8 normal brain tissues were negative for Tag. Tag-p53 complex was detected in all of 20 Tag positive tumors as well as Tag-Rb complex in all of 15 Tag positive tumors. Conclusion: SV40 Tag is not only expressed in human brain tumors, but also it can form specific complexes with tumor suppressors p53 and Rb. SV40 is correlated to human brain tumorigenesis. The inactivation of p53 and Rb due to the formation of Tag-p53 and Tag-Rb complexes is possibly an important mechanism in the etiopathogenesis of human brain tumors.

Establishment of a Tumor-bearing Mouse Model Stably Expressing EGFP Labeled Human MUC1 VNTRs

Two eukaryotic vectors expressing 9 tandem repeats of human MUCI（VNTR）, VR1012-VNTR, and pEGFP-VNTR, were constructed by cloning VNTR gene into VR1012 and pEGFP, respectively. VNTR stably expressing murine Lewis lung carcinoma（LLC） cell line（VNTR^＋ LLC） was established by Lipofectamine-mediated transfection of pEGFP-VNTR into LLC cells. The EGFP expression was observed under a fluorescent microscope and VNTR expression in VNTR^＋ LLC cells was confirmed by means of Western blotting. A syngenic graft tumor model was generated by subcutaneous injection of VNTR^＋ LLC cells into C57/BL6 mice and tumor size increased rapidly with time and in a cell qumber dependent manner. VNTR mRNA expression in the tumor formed was confirmed by RT-PCR. After the third immunization mice were challenged subcutaneously with 5×10^5 VNTR^＋ LLC cells, a significant reduction of subcutaneous tumor growth was observed in the groups immunized with VNTR plasmid DNA compared with that in the groups immunized with the vector DNA alone. Thus, the suppression of subcutaneous tumor was antigen-specific. This model is useful for the development of tumor vaccines targeting MUCI VNTRs.

HCA520, A NOVEL TUMOR ASSOCIATED ANTIGEN, INVOLVED IN CELL PROLIFERATION AND APOPTOSIS

Objective: Tumor associated antigen encoding gene HCA520 (AF146019) was identified by screening a human hepatocellular carcinoma expressing cDNA library using SEREX technique. In this experiment we studied the effect of HCA520 on cell proliferation and apoptosis. Methods: Gene HCA520 was gained by PCR and transfected into 293 cells. The stable expression cells were obtained by G418 selection. The cell proliferation was measured by [3H]-TdR uptake and apoptosis assay was measured by FACS. Results: Eukaryotic expression plasmid pcDNA3-HCA520 was constructed and its stable transfectants were obtained. Overexpression of HCA520 inhibited the cell proliferation and enhanced cell apoptosis after serum deprivation. Conclusion: HCA520 is a novel tumor associated antigen that can affect cell proliferation and apoptosis.

肝胆肿瘤中肿瘤特异性CircRNA衍生抗原肽的鉴定

基于肿瘤抗原的免疫治疗的应用受到验证免疫原性肽稀缺性的阻碍。本研究旨在研究环状RNA(circRNA)在肝胆肿瘤类器官中作为肿瘤抗原肽新来源的潜力。使用RNA测序(RNA-seq)和基于算法的评分工具,预测3950个翻译的肿瘤特异性环状RNA在27个类器官中产生18971个抗原肽。从抗原格局来看,11个氨基酸长度(mer)肽和人白细胞抗原(HLA)-A结合肽具有最高的免疫原性相关评分。在分析的3/5类器官中,有13个预测抗原肽通过质谱(MS)免疫肽组学被直接确认为HLA-A、HLA-B和HLA-C(HLA-ABC)结合肽。在流式细胞术和酶联免疫吸附试验(ELISA)中,由HLA-ABC分子呈递的circRNA衍生的肿瘤特异性肽刺激CD8(CD8)T细胞,显示CD107a干扰素γ(IFNγ)共表达和IFNγ分泌增加。免疫原性环状RNA衍生肽诱导的靶向类器官的细胞毒性T细胞活性在杀伤实验中得到验证。值得注意的是,来自circTBC1D15的抗原肽YGFNEILKK不仅被认为是类器官的HLA-ABC呈递肽,而且还显著降低了肿瘤类器官的存活率。本研究的发现强调了产生肿瘤抗原的一个关键亚群,这对靶向肿瘤特异性circRNA具有重要意义。

Personalized pancreatic cancer therapy:from the perspective of mRNA vaccine

Pancreatic cancer is characterized by inter-tumoral and intra-tumoral heterogeneity,especially in genetic alteration and microenvironment.Conventional therapeutic strategies for pancreatic cancer usually suffer resistance,highlighting the necessity for personalized precise treatment.Cancer vaccines have become promising alternatives for pancreatic cancer treatment because of their multifaceted advantages including multiple targeting,minimal nonspecific effects,broad therapeutic window,low toxicity,and induction of persistent immunological memory.Multiple conventional vaccines based on the cells,microorganisms,exosomes,proteins,peptides,or DNA against pancreatic cancer have been developed;however,their overall efficacy remains unsatisfactory.Compared with these vaccine modalities,messager RNA(mRNA)-based vaccines offer technical and conceptional advances in personalized precise treatment,and thus represent a potentially cutting-edge option in novel therapeutic approaches for pancreatic cancer.This review summarizes the current progress on pancreatic cancer vaccines,highlights the superiority of mRNA vaccines over other conventional vaccines,and proposes the viable tactic for designing and applying personalized mRNA vaccines for the precise treatment of pancreatic cancer.

mRNA vaccine development for cholangiocarcinoma:a precise pipeline

Cholangiocarcinoma(CHOL)is one of the most aggressive tumors worldwide and cannot be effectively treated by conventional and novel treatments,including immune checkpoint blockade therapy.The mRNA vaccine-based immunotherapeutic strategy has attracted much attention for various diseases,however,its application in CHOL is limited due to the thoughtlessness in the integration of vaccine design and patient selection.A recent study established an integrated path for identifying potent CHOL antigens for mRNA vaccine development and a precise stratification for identifying CHOL patients who can benefit from the mRNA vaccines.In spite of a promising prospect,further investigations should identify immunogenic antigens and onco-immunological characteristics of CHOL to guide the clinical application of CHOL mRNA vaccines in the future.

Cancer/testis antigen, Kita-Kyushu lung cancer antigen-1 and ABCD stratification for diagnosing gastric cancers

BACKGROUND The ABCD stratification[combination of serum pepsinogen(PG)levels and titers of antibody(immunoglobulin G,IgG)against Helicobacter pylori(H.pylori)]is effective for the classification of individuals at risk of developing gastric cancer(GC).The Kita–Kyushu lung cancer antigen-1(KK-LC-1)is a Cancer/Testis antigen frequently expressed in GC.AIM To evaluate the effectiveness of KK-LC-1 and ABCD stratification in the diagnosis of GC.METHODS We analyzed the gene expression of KK-LC-1 in surgical specimens obtained from GC tumors.The levels of serum PG I/PG II and IgG against H.pylori were measured.According to their serological status,the patients were classified into the four groups of the ABCD stratification.RESULTS Of the 77 examined patients,63(81.8%)expressed KK-LC-1.The IgG titers of H.pylori and PG II were significantly higher in patients expressing KK-LC-1 than those measured in patients not expressing KK-LC-1(P=0.0289 and P=0.0041,respectively).The expression of KK-LC-1 in group C[PG method(+)/H.pylori infection(+)]was as high as 93.9%high.KK-LC-1 was also detected in group A[-/-].CONCLUSION The KK-LC-1 expression in GC was associated with H.pylori infection and atrophic status,so that,KK-LC-1 may be a useful marker for the diagnosis of GC.

Bacterial outer membrane vesicle-based cancer nanovaccines

Tumor vaccines,a type of personalized tumor immunotherapy,have developed rapidly in recent decades.These vaccines evoke tumor antigen-specific T cells to achieve immune recognition and killing of tumor cells.Because the immunogenicity of tumor antigens alone is insufficient,immune adjuvants and nanocarriers are often required to enhance anti-tumor immune responses.At present,vaccine carrier development often integrates nanocarriers and immune adjuvants.Among them,outer membrane vesicles(OMVs)are receiving increasing attention as a delivery platform for tumor vaccines.OMVs are natural nanovesicles derived from Gramnegative bacteria,which have adjuvant function because they contain pathogen associated molecular patterns.Importantly,OMVs can be functionally modified by genetic engineering of bacteria,thus laying a foundation for applications as a delivery platform for tumor nanovaccines.This review summarizes 5 aspects of recent progress in,and future development of,OMV-based tumor nanovaccines:strain selection,heterogeneity,tumor antigen loading,immunogenicity and safety,and mass production of OMVs.

SIGNIFICANCE OF CEA AND CA242 IN THE DIAGNOSIS OF COLORECTAL CARCINOMA

Carcinoembryonic antigen (CEA) is frequently used in the diagnosis of the colorectal carcinoma. CA242 is a novel unique tumor-associated antigen characterized by higher tumor specificity and sensitivity for colorectal cancer, as compared with other mucin antigens. In this study, preoperative levels of serum CEA and CA242 were measured in 63 cases of colorectal carcinoma. It was disclosed that the positive rate of CA242 was higher than that of CEA, particularly in patients with colon cancer.The combined determination of CEA and CA242 significantly increased the sensitivity and accuracy in the detection of colorectal cancer as compared with the use of CEA alone (P<0.o1). In patients with advanced disease the positive rate was markedly elevated, especially in patients with liver metastasis. The results indicate that the combined use of CEA and CA242 assays is an useful adjunct diagnostic measure for colorectal carcinoma, and is helpful in the assessment of the stage of the disease as well as in making treatment Plan.

The development and functions of CD41 T cells expressing a transgenic TCR specific for an MHC-I-restricted tumor antigenic epitope

It has been reported that the ratio of CD41 to CD81 T cells has no bias in a few class I major histocompatibility complex(MHC-I)-restricted T-cell receptor(TCR)-transgenic mice specific for alloantigens or autoantigens,in which most CD41 T cells express an MHC-I-restricted TCR.In this study,we further showed that more than 50%of CD41 T cells in MHC-I-restricted P1A tumor antigen-specific TCR(P1ATCR)-transgenic mice could specifically bind to MHC-I/P1A peptide complex.P1A peptide could stimulate the transgenic CD41 T cells to proliferate and secrete both type 1 helper T cell and type 2 helper T cell cytokines.The activated CD41 T cells also showed cytotoxicity against P1A-expressing tumor cells.The analysis of TCR a-chains showed that these CD41 T cells were selected by co-expressing endogenous TCRs.Our results show that CD41 T cells from P1ATCR transgenic mice co-expressed an MHC-I-restricted transgenic TCR and another rearranged endogenous TCRs,both of which were functional.

Developing effective tumor vaccines: basis, challenges and perspectives

A remarkable advance in tumor immunology during the last decade is the elucidation of the antigenic basis of tumor recognition and destruction.A variety of tumor antigens have been identified using several strategies including conventional experiments and newly developed bioinformatics.Among these antigens,cancer/testis antigen(CT antigen)is considered to be the most promising target for immunotherapy by vaccination.Successful immunotherapy of tumors requires understanding of the natural relationship between the immune system and tumor in the status of differentiation,invasion and maturation.Continued progress in development of effective cancer vaccines depends on the identification of appropriate target antigens,the establishment of optimal immunization strategies without harmful autoimmune responses and the ability of manipulating tumor microenvironment to circumvent immune suppression and to augment the anti-tumor immune response.

A Novel Putative Tumor Associated Antigen

A is a tumor associated antigen. Monoclonal antibody (mAb) against 17 1A has been used in adjuvant therapy of colorectal carcinoma. Using mAb against 17 1A antigen on affinity chromatography, a novel putative tumor associated antigen (P50) whose relative molecular mass is 5 0×10 4 has been isolated from human colorectal tumor tissues which are recognized by mAbs 17 1A and M79, while the relative molecular mass of 17\|1A antigen isolated from several colorectal tumor cell lines is 3 3×10 4. P50 was recognized by mAbs 17 1A and M79 which are specific mAbs against 17 1A antigen.

PROSPECTIVE STUDY OF MULTIPLE GENETIC TUMOR MARKER ASSAY BY QUANTITATIVE REAL-TIME PCR TO PREDICT RECURRENCE IN COLORECTAL CANCER PATIENTS

Objective To describe correlation between multiple genetic tumor markers,carcinoembryonic antigen (CEA),cytokeratin 20 (CK20),and Survivin,and clinicopathological features of colorectal cancer (CRC) and to assess prognostic diagnosis value in cancer recurrence and metastasis.Methods A total of 92 patients with CRC,68 patients with precancerous lesions,and 29 control volunteers were collected for the detection of CEA,CK20,and Survivin expressions by using quantitative Real-Time PCR technology.Associations among these measurements and clinicopathological features of CRC,and cancer recurrence and metastasis rates in 4-year follow-up were analyzed.Results No mRNA expressions of CEA,CK20,or Survivin were detected in the control group.Expressions of CEA,CK20,and Survivin were 41.3%,47.8%,and 72.8% in CRC patients,respectively.The expressions of genetic tumor markers were related to the clinical stage and lymph node metastasis.In patients with Survivin high expression,4-year survival rate was significantly lower than that in Survivin low expression.The multiple tumor markers assay for CRC patients showed higher specificity and positive detection rate than single marker assay.Patients with CEA,CK20,and Survivin simultaneous expressions had significantly higher 4-year recurrence rate and death rate than those with only one or two markers expression.ConclusionMultiple tumor markers assay including CEA,CK20,and Survivin in peripheral blood by quantitative Real-Time PCR can be an ideal method for the surveillance of the recurrence and prognosis for CRC patients.

Clinical significance of the loss of CD20 antigen on tumor cells in patients with relapsed or refractory follicular lymphoma

Aim:Anti-CD20 monoclonal antibody is a cornerstone therapy for follicular lymphoma.Following anti-CD20 therapy,a potential decrease in CD20 antigen,and therefore a loss of the tumor target might be expected.However,the incidence and clinical significance of CD20 loss on tumor cells in patients with relapsed or refractory follicular lymphoma are unknown.This study aims to investigate the incidence and outcome of patients with relapsed or refractory follicular lymphoma patients harboring the loss of the tumor target,CD20.Methods:All consecutive adult patients with relapsed or refractory follicular lymphoma referred to the Early Drug Department at Gustave Roussy were included.The main objectives were to assess the incidence and prognosis of the loss in expression of CD20 antigen on the surface of tumor cells on patient outcome.Results:Over the study period 2013-2018,131 patients were screened for clinical trials with B-cell malignancies in the early drug department of Gustave Roussy in France.Forty-four patients presented with relapsed or refractory follicular lymphoma and 32 had tumor biopsies at the time of relapse that were retained for analysis.The median(range)age was 67.5 years(55.3-75.3)and the median number of prior anti-cancer systemic therapies was 3(2-4).At the time of relapse,CD20 expression was positive in 84%of tumors(n=27)and negative in 16%of tumors(n=5).At a median follow-up of 18.3(0.6-83.3)months,CD20 negativity was associated with a poorer prognosis with a median overall survival of 8.9 months(95%CI:2.4-19.1)in comparison to CD20 positive patients(28.3 months,95%CI:25.1-75.3 months,P=0.019).Conclusion:The loss of the tumor target antigen,CD20,occurred in 16%of patients with relapse or refractory follicular lymphoma.Due to confounding factors in patients who received anti-CD20 immunotherapy,it was not possible to formally establish the prognostic significance of CD20 negativity.However,we suggest that a check for CD20 antigen positivity nevertheless be performed to adapt subsequent therapies for patients with relapsed or refractory follicular lymphoma.