Pan-TRK positive uterine sarcoma in immunohistochemistry without neurotrophic tyrosine receptor kinase gene fusions:A case report

BACKGROUND The classification of uterine sarcomas is based on distinctive morphological and immunophenotypic characteristics,increasingly supported by molecular genetic diagnostics.Data on neurotrophic tyrosine receptor kinase(NTRK)gene fusionpositive uterine sarcoma,potentially aggressive and morphologically similar to fibrosarcoma,are limited due to its recent recognition.Pan-TRK immunohistochemistry(IHC)analysis serves as an effective screening tool with high sensitivity and specificity for NTRK-fusion malignancies.CASE SUMMARY We report a case of a malignant mesenchymal tumor originating from the uterine cervix,which was pan-TRK IHC-positive but lacked NTRK gene fusions,accompanied by a brief literature review.A 55-year-old woman presented to the emergency department with abdominal pain and distension,exhibiting significant ascites and multiple solid pelvic masses.Pelvic examination revealed a tumor encompassing the uterine cervix,extending to the vagina and uterine corpus.A punch biopsy of the cervix indicated NTRK sarcoma with positive immunochemical pan-TRK stain.However,subsequent next generation sequencing revealed no NTRK gene fusion,leading to a diagnosis of poorly differentiated,advanced-stage sarcoma.CONCLUSION The clinical significance of NTRK gene fusion lies in potential treatment with TRK inhibitors for positive sarcomas.Identifying such rare tumors is crucial due to the potential applicability of tropomyosin receptor kinase inhibitor treatment.

Profiling of gene fusion involving targetable genes in Chinese gastric cancer

BACKGROUND Approximately half of all new cases of gastric cancer(GC)and related deaths occur in China.More than 80%of patients with GC are diagnosed at an advanced stage,which results in poor prognosis.Although HER2-directed therapy and immune checkpoint inhibitors have been somewhat successful,new drugs are still needed for the treatment of GC.Notably,several gene fusion-targeted drugs have been approved by the United States Food and Drug Administration for solid tumors,including GC,such as larotrectinib for NTRK fusion-positive cancers and zenocutuzumab for NRG1 fusion-positive cancers.However,gene fusions involving targetable genes have not been well characterized in Chinese patients with GC.AIM To identify the profile of fusions involving targetable genes in Chinese patients with GC using clinical specimens and determine the distribution of patients with gene fusion variants among the molecular subtypes of GC.METHODS We retrospectively analyzed gene fusion events in tumor tissue samples from 954 Chinese patients with GC.Clinicopathological characteristics were obtained from their medical records.Genetic alterations,such as single nucleotide variants,indels,amplifications,and gene fusions,were identified using a targeted sequencing panel containing 825 genes.Fusions were validated by fluorescence in situ hybridization(FISH)using break-apart probes.The microsatellite instability(MSI)status was evaluated using MSIsensor from the targeted sequencing panel data.Tumor mutational burden(TMB)was calculated using the total number of nonsynonymous mutations divided by the total genomic targeted region.Chi-square analysis was used to determine the enrichment of gene fusions associated with the molecular subtypes of GC.RESULTS We found that 1.68%(16/954)of patients harbored 20 fusion events involving targetable genes.RARA fusions(n=5)were the most common,followed by FGFR2,BRAF,MET,FGFR3,RET,ALK,EGFR,NTRK2,and NRG1 fusions.Two of the RARA fusions,EML4-ALK(E6:E20)and EGFRSEPTIN14(E7:E10),have been identified in other tumors but not in GC.Surprisingly,18 gene fusion events were previously not reported in any cancer types.Twelve of the eighteen novel gene fusions included complete exons encoding functional domains of targetable genes,such as the tyrosine kinase domain of receptor tyrosine kinases and the DNA-and ligand-binding domains of RARA.Consistent with the results of detection using the targeted sequencing fusion panel,the results of FISH(fluorescence in situ hybridization)confirmed the rearrangement of FGFR2 and BRAF in tumors from patients 04 and 09,respectively.Genetic analysis indicated that the fusion genes were significantly enriched in patients with ERBB2 amplification(P=0.02);however,there were no significant differences between fusion-positive and fusion-negative patients in age,sex,MSI status,and TMB.CONCLUSION We characterized the landscape of fusions involving targetable genes in a Chinese GC cohort and found that 1.68%of patients with GC harbor potential targetable gene fusions,which were enriched in patients with ERBB2 amplification.Gene fusion detection may provide a potential treatment strategy for patients with GC with disease progression following standard therapy.

The Construction of Fusion Expression Vector Carrying GFP and TP of GGPPS from Ginkgo biloba L.

[Objective]The aim was to construct the fusion gene expression vector which consisted of GFP and TP gene of GGPPS from the Ginkgo biloba L.[Method]The transit-peptide（TP） sequence of GGPPS from cDNA of Ginkgo biloba L.was successfully cloned by using DNA recombination technology,which was then linked to the efficient plant expression vector p1304 ＋ to construct the fusion gene expression vector p1304 ＋-TP.Then engineering strain EHA105-p1304 ＋-TP was constructed by transformed p1304 ＋-TP to Agrobacterium rhizogenes EHA105 using freeze-thaw method.[Result]The fusion gene expression vector which consisted of GFP and TP gene of GGPPS from the Ginkgo biloba L.and engineering strain EHA105-p1304 ＋-TP were successfully constructed.[Conclusion]It lays a foundation for further study of subcellular localization of TP transit peptide,which can help to clarify the molecular mechanism of a key step in biosynthesis of ginkgolides precursors,and also provides an important basis for the research on metabolic engineering of ginkgolide.

Detection of the SYT-SSX Fusion Gene in Synovial Sarcoma

Objective: To investigate the feasibility and significance of detecting SYT-SSX fusion gene in paraffin-embedded tissues of synovial sarcoma (SS) by reverse-transcriptase polymerase chain reaction(RT-PCR) methods. Methods: Twenty cases of SS tumors from archival materials were collected and all samples were formalin-fixed and paraffin-embedded (FFPE). SYT-SSX fusion transcript was detected by RT-PCR. Home-keeping gene Porphobilinogen Deaminase (PBGD) was regarded as internal control.Results: PBGD mRNA was detected in all 20 tumor cases (100%). SYT-SSX fusion transcript was detected in 18 tumor cases (90%). In 18 SYT-SSX positive SS cases, there are 12 present SYT-SSX1 fusion transcript and 6 present SYT-SSX2 fusion transcript. SYT-SSX1 fusion transcript can be seen in 9 monophasic SS and 3 biphasic SS. In 6 SYT-SSX2 positive SS cases, 4 were monophasic SS and 2 were biphasic. Conclusion: Detection of SYT-SSX fusion transcripts in FFPE tissues for diagnosis of SS is feasible and sensitive. Subtypes of SYT-SSX fusion gene may provide prognosis information.

Left lower lobe sleeve resection for the clear cell variant of pulmonary mucoepidermoid carcinoma:A case report

BACKGROUND Pulmonary mucoepidermoid carcinoma(PMEC)is a rare malignancy that arises from minor salivary glands within the tracheobronchial tree.The clear cell variant of PMEC is exceptionally uncommon and presents notable diagnostic challenges,primarily attributable to its morphological similarity to other tumors containing clear cells.CASE SUMMARY A 22-year-old male,formerly in good health,came in with a two-month duration of persistent cough and production of sputum.Subsequent imaging and bronchoscopy examinations revealed a 2 cm tumor in the distal left main bronchus,which resulted in complete atelectasis of the left lung.Further assessment via positron emission tomography/computed tomography scans and endoscopic biopsy confirmed the primary malignant nature of the tumor,charac-terized by clear cell morphology in most of the tumor cells.The patient underwent a left lower lobe sleeve resection accompanied by systematic mediastinal lymph node dissection.Molecular pathology analysis subsequently revealed a CRTC3-MAML2 gene fusion,leading to a definitive pathological diagnosis of the clear cell variant of PMEC,staged as T2N0M0.After surgery,the patient experienced a smooth recovery and exhibited no signs of recurrence during the one-and-a-half-year follow-up period.CONCLUSION This article describes an unusual case of a clear cell variant of PMEC characterized by the presence of a CRTC3-MAML2 gene fusion in a 22-year-old male.The patient underwent successful left lower lobe sleeve resection.This case underscores the distinctive challenges associated with diagnosing and treating this uncommon malignancy,underscoring the importance of precise diagnosis and personalized treatment strategies.

Transgenic rice expressing a novel phytase-lactoferricin fusion gene to improve phosphorus availability and antibacterial activity

The developing trends of livestock production are efficiency availability of phytate phosphorus and abuse of antibiotics. safety and sustainability, which face two major challenges： low As a solution phytases and antimicrobial peptides are applied as feed additives. However, phytases and antimicrobial peptides are susceptible to proteases, costly by fermentation and potential toxic to production hosts. We transformed an optimized phytase-lactoferricin fusion gene PhyLfdriven by an en- dosperm-specific promoter Gt13aP and Bar （bialaphos resistance） gene as a selection maker into rice. The Bar and PhyLf genes were integrated into the rice genome, stably inherited and expressed. Their phosphinothricin acetyl transferase （PAT） protein content oftransgenic plants with glufosinate resistance varied between 50.45-93.39 IJg g-l. Fusion protein expressed especially in the seeds of transgenic rice had a summit phytase activity at 32.30 U g-l, which increased by 61.71-fold com- pared to the control/check group （CK） and 7.54-fold compared to un-optimized transgenic plant. The highest inorganic phosphorus （Pi） content of the transgenic seeds reached 13.15 mg g-~, increased by 12.77-fold compared to that of CK. Preliminary antibacterial experiments showed that the enterokinase hydrolysate product of fusion protein could inhibit the growth of Escherichia coil DH5a. These results indicated that the protein PhyLf has the potential to increase availability of feed phytate phosphorus, improve consumer＇s immunity and reduce the use of antibiotics.

Cloning of α-β fusion gene from Clostridium perfringens and its expression

AIM： To study the cloning of α-β fusion gene from Closindium perfringens and the immunogenidty of 0-6 fusion expression. METHODS： Cloning was accomplished after PCR amplification from strains NCTC64609 and C58-1 of the protective antigen genes of α-toxin and β-toxin. The fragment of the gene was cloned using plasmid pZCPAB. This fragment coded for the gene with the stable expression of α-β fusion gene binding. In order to verify the exact location of the α-β fusion gene, domain plasmids were constructed. The two genes were fused into expression vector pBV221. The expressed α-β fusion protein was identified by ELISA, SDS-PAGE, Western blotting and neutralization assay. RESULTS： The protective co-toxin gene （cpa906） and the β-toxin gene （cpb930） were obtained. The recombinant plasmid pZCPAB carrying α-β fusion gene was constructed and transformed into BL21（DE3）. The recombinant strain BL21（DE3）（pZCPAB） was obtained. After the recombinant strain BL21（DE3）（pZCPAB） was induced by 42℃, its expressed product was about 22.14% of total cellular protein at SDS-PAGE and thin-layer gel scanning analysis. Neutralization assay indicated that the antibody induced by immunization with α-βfusion protein could neutralize the toxicity of α-toxin and β-toxin. CONCLUSION： The obtained α-toxin and β-toxin genes are correct. The recombinant strain BL21（DE3）（pZCPAB） could produce α-β fusion protein. This protein can be used for immunization and is immunogenic. The antibody induced by immunization with α-β fusion protein could neutralize the toxicity of α-toxin and β-toxin.

PCA3 and TMPRSS2-ERG gene fusions as diagnostic biomarkers for prostate cancer

The incidence of prostate cancer （PCa） is rising steadily among males in many countries. Serum prostate-specific antigen （PSA） is widely applied to clinical diagnosis and screening of PCa. However, the so-called grey area of PSA levels 4.0-10.0 ng/mL has a low specificity of 25-40% resulting in a high rate of negative biopsy and overtreatment. So in order to treat PCa patients in early stage, there is an urgent need for new biomarkers in PCa diagnosis. The PCA3 gene, a non-coding RNA （ncRNA） that is highly expressed in prostate cancer （PCa） cells, has been identified as a molecular biomarkers to detect PCa, of which PCA3 has already under clinical application. PCA3 is strongly overexpressed in malignant prostate tissue compared to benign or normal adjacent one. Newly, PCA3 is considered to be a promising biomarker in clinical diagnosis and targeted therapy. The diagnostic significance of PCA3, however, is awaiting further researches. Moreover, it has been demonstrated recently that TMPRSS2-ERG gene fusion is identified as the predominant genetic change in patients diagnosed with PCa. Recent study revealed that combination of the PC43 and TMPRSS2-ERG gene fusion test optimizes PCa detection compared with that of single biomarker, which would lead to a considerable reduction of the number of prostate biopsies. In this review, we focused on the potential use of PCA3 and TMPRSS2-ERG gene fusion detection in the diagnosis of PCa.

Immunogenicity and protective efficacy of DHBV DNA vaccines expressing envelope and capsid fusion proteins in ducks delivered by attenuated Salmonella typhimurium

Duck hepatitis B virus(DHBV) shares many basic characteristics with hepatitis B virus(HBV) and is an attractive model for vaccine development. In this study, DHBV DNA vaccines were designed to express envelope and capsid fusion proteins to enhance the breadth of immune response in ducks. Attenuated Salmonella typhimurium(SL7207) was used as a carrier and adjuvant to boost the magnitude of immune response. Based on this strategy, novel DNA vaccines(SL7207-p VAX1-LC and SL7207-p VAX1-SC) were generated. Growth kinetics, genetic stabilities and relative transcription levels of the L, S and C genes introduced by these vaccine strains were measured before inoculation to guarantee safety and efficacy. The relative transcript levels of the CD4 and CD8 T genes and the antibody levels(Ig Y) in ducks receiving the vaccines were higher than those in single gene delivered groups. Additionally, the copy number of covalently closed circular DNA in hepatocytes after DHBV challenge also provided evidence that our fusion vaccines could enhance the protective efficiency against DHBV infection in ducks.

Stable Expression of Hantavirus H8205 Strain G1/IL-2 Gene and Immune Protection of the Fusion Gene

To explore the feasibility of stable expression of Hantavirus H8205 strain G1 segment and human IL-2 fusion gene in Vero cells, and to examine the immune protection effects on mice vaccinated with this recombinant eukaryotic expression vector containing Hantavirus G1 gene and IL-2 gene. With the help of lipofectamine, the Vero cells were transfected with pcDNA3.1/HisB-IL-2-G1 and the positive cells were selected by G418. IFAT and SDS-PAGE elec- trophoresis were used to determine the stable transfection and expression of recombinant protein. Each mouse was inoculated with plasmids intramuscularly （i.m.） three times, 2 boosts were given at 2-week intervals, serum anti-hantavirus antibodies were detected by ELISA and neutralizing antibodies （NAb） were detected by Plaque Reduction Neutralization Test. The fusion protein expressed in Vero cells was 78 kD, corresponding to the estimated molecular size. The neutralizing antibody titers of mice with pcDNA3.1/HisB-IL-2-G1 were 1：20-1：80. IL-2/G1 fusion gene could be transferred in Vero cells and stably express the fusion protein. Specific humeral immune responses in mice can be induced with the recombinant eukaryotic expression vector containing the fusion gene, which lays the foundation for further development of therapeutic HTNV vaccine.

Detection of TMPRSS2：ERG fusion gene in circulating prostate cancer cells

Aim： To investigate the existence of TMPRSS2：ERG fusion gene in circulating tumor cells （CTC） from prostate cancer patients and its potential in monitoring tumor metastasis. Methods- We analyzed the frequency of TMPRSS2： ERG and TMPRSS2：ETV1 transcripts in 27 prostate cancer biopsies from prostatectomies, and TMPRSS2：ERG transcripts in CTC isolated from 15 patients with advanced androgen independent disease using reverse transcription polymerase chain reaction （RT-PCR）. Fluorescence in situ hybridization （FISH） was applied to analyze the genomic truncation of ERG, which is the result of TMPRSS2：ERG fusion in 10 of the 15 CTC samples. Results： TMPRSS2： ERG transcripts were found in 44% of our samples, but we did not detect expression of TMPRSS2：ETV1. Using FISH analysis we detected chromosomal rearrangements affecting the ERG gene in 6 of 10 CTC samples, including 1 case with associated TMPRSS2：ERG fusion at the primary site. However, TMPRSS2：ERG transcripts were not detected in any of the 15 CTC samples, including the 10 cases analyzed by FISH. Conclusion： Although further study is required to address the association between TMPRSS2：ERG fusion and prostate cancer metastasis, detection of genomic truncation of the ERG gene by FISH analysis could be useful for monitoring the appearance of CTC and the potential for prostate cancer metastasis.

INHIBITION OF APOPTOSIS BY bcr-abl FUSION GENE IN K562 CELLS

Objective: To investigate the effect of bcr-abl fusion gene on CML cell apoptosis. Methods: Apoptosis of ex-vivo cultured K562 cells were observed after exposure to synthetic 18 mer antisense oligodeoxynucleotide complementary to the bcr-abl junction (b3a2). Results: Apoptosis of K562 cells was significantly increased associated with inhibition of bcr-abl expression. Conclusion: bcr-abl fusion gene formation due to chromosome translocation may be the major mechanism of CML via inhibition of apoptosis.

APOPTOSIS INDUCTION BY THE RECOMBINANT FUSION APOPTOSIS INDUCING FACTOR ON HELA CELLS

Objective: To obtain the recombinant fusion AIF genes inserted into the eukaryotic expression vectorpIRES2-EGFP, to observe the expression and location ofthe fusion AIF genes (3NE: PE(280-358)-AIF1-120, and4NE: PE(280-364)-AIF1-120), and to detect and compare their apoptosis inducing effects on the transfected HeLacells. Methods: Full-length human AIF gene was clonedby RT-PCR, and its N-terminal mitochondrial localization sequence (MLS) was replaced by part sequence ofPsuedomonas exotoxin A (PE) translocation domain(PEII(280-358/364)), then the recombinant fusion geneswere inserted into the pIRES2-EGFP eukaryotic expression vector. After these genes were transiently transfected into HeLa cells with LipofectAmine, the expression of therecombinant fusion AIF genes and their effects on HeLacells were detected by fluorescent microscopy, laser confocal microscopy and electron microscopy. Results: Theeukaryotic expression vectors containing the recombinant fusion AIF genes (pIRES2-EGFP-PEII(280-358/364)-AIF1- 120) were constructed successfully. It wasdemonstrated that the fusion AIF protein genes wereexpressed effectively in the transfected cells, with the GFP co-expressed in cells by indirect immunofluorescence staining analysis. After transfection, expression of the genes could induce HeLa cells to exhibit the typical apoptosis features: such as plasma membrane blebbing and peripheral chromatin condensation. As compared with control groups, the untreated cells and the void vector transfected cells, the living cell number of the AIF gene transfected cells reduced distinctly. Conclusion: Our data prove that the expression of the recombinant human AIF fusion genes could induce apoptosis in transfected HeLa cells, which provides new strategy for cancer killing.

VISUALIZATION OF HEAD AND NECK CANCER MODELS WITH A TRIPLE FUSION REPORTER GENE

The development of experimental animal models for head and neck tumors generally rely on the biol uminescence imaging to achieve the dynamic monitoring of the tumor growth and metastasis due to the complicated anatomical structures.Since the bioluminescence imaging is largely affected by the intracellular luciferase expression level and external D-luciferin concentrations,its imaging accuracy requires further confirmation.Here,a new triple fusion reportelr gene,which consists of a herpes simplex virus type 1 thymidine kinase(TK)gene for radioactive imaging,a far-red fuorescent protein(mLumin)gene for fuorescent imaging,and a firefly luciferase gene for bioluminescence imaging,was introduced for in vrivo observation of the head and neck tumors through multi-modality imaging.Results show that fuorescence and bioluminescence signals from mLumin and luciferase,respectively,were clearly observed in tumor cells,and TK could activate suicide pathway of the cells in the presence of nucleotide analog-ganciclovir(GCV),demonstrating the effecti veness of individual functions of each gene.Moreover,subcutaneous and metastasis animal models for head and neck tumors using the fusion reporter gene-expressing cell lines were established,allowing multi-modality imaging in vio.Together,the established tumor models of head and neck cancer based on the newly developed triple fusion reporter gene are ideal for monitoring tumor growth,assessing the drug therapeutic efficacy and verifying the effec-tiveness of new treatments.

Construction of Fusion Expression Vector Carrying GFP and ZmCIPK

[Objective] The aim was to isolate the CBL-interacting protein kinases(CIPK)from maize(Zea mays L.)and construct the fusion gene expression vector which consisted the ZmCIPK8 and GFP.[Method] The ZmCIPK8 cDNA was successfully cloned by using RT-PCR method.And then,it was connected to the pBlueScript SK(pSK)plasmid,which contained the GFP gene.So that the fusion gene vector pSK-CIPK-GFP was obtained.Then,the fusion gene was connected into the efficient plant expression vector PBI121 to construct the fusion gene expression vector PBI-CIPK-GFP.At last,the recombined expression vector was transformed to Agrobacterium tumefaciems LBA4404 to produce the engineering strain LBA4404-PBI-CIPK-GFP.[Result] The fusion gene expression vector which consisted of GFP and ZmCIPK8 gene and engineering strain LBA4404-PBI-CIPK-GFP were successfully constructed.[Conclusion] The results lays a foundation for further study of subcellular localization of ZmCIPK8,which can help to clarify the molecular mechanism of regulation serious stresses,and also provides an important basis for the research on resistance stress engineering of maize.

Detection of EGFR-SEPT14 fusion in cell-free DNA of a patient with advanced gastric cancer: A case report

BACKGROUND Gastric cancer is the fifth most diagnosed cancer worldwide and the third most common cause of cancer-related death.In recent decades,increasing application of next-generation sequencing has enabled detection of molecular aberrations,including fusions.In cases where tissue is difficult to obtain,cell-free DNA(cfDNA)is used for detecting mutations to identify the molecular profile of cancer.Here,we report a rare case of EGFR-SEPT14 fusion detected from cfDNA analysis in a patient with gastric cancer.CASE SUMMARY A 49-year-old female diagnosed with advanced gastric cancer in July 2019 received capecitabine and then combination chemotherapy of ramucirumab and paclitaxel,but ascites was detected.The therapy was switched to nivolumab,but disease progression was observed on a positron emission tomography/computed tomography scan in May 2020.Therapy was discontinued,and cfDNA nextgeneration sequencing was immediately evaluated.All genomic variants,including fusions,were analyzed from cfDNA.The following somatic alterations were detected from the patient’s cfDNA:an APC frameshift mutation(NM_000038.5:c.6579del,p.V2194fs)with variant allele frequency of 0.5%,an EGFR amplification with a copy number of 17.3,and an EGFR-SEPT14 fusion with variant allele frequency of 45.3%.The site of the fusion was exon 24 of EGFR fused to exon 10 of SEPT14.The fusion was in-frame and considered to be protooncogenic. Although the patient refused to continue therapy, we suggest thatEGFR-targeted therapies be tried in such future cases.CONCLUSIONThe expanded applications of the cfDNA assay may open a new horizon intreatment of patients with advanced gastric cancer.

Detection of EWS-FLI1 fusion transcripts in paraffin embedded tissues of peripheral primitive neuroectodermal tumors by nested reverse transcription polymerase chain reaction

Objective: To assess the feasibility and significance of detecting EWS-FLI1fusion transcripts in paraffin embedded tissues of peripheral primitive neuroectodermal tumors (PNETs) by nested reverse transcription polymerase chain reaction(RT-PCR). Methods: Twelve formalin-fixed and paraffin-embedded (FFPE) samples of PNET were retrieved from archive and consultation materials, together with eight cases of controlled tumor. EWS-FLI1 fusion transcripts were detected by nested RT-PCR. Home-keeping gene β-actin was used to detect the quality of mRNA. Results: β-actin mRNA was detected in 9 of the 12 tumor cases. EWS-FLI1 fusion transcripts were detected in 6 cases, among which 4 had a “type 1” fusion transcript and 2 had a “type 2” fusion transcript. None of the controlled tumor was detected the fusion gene. Conclusion: RT-PCR is a feasible method for the detection of EWS-FLI1 fusion transcripts in FFPE tissues in PNET and the result is meaningful in differential diagnosis and prognostic evaluation.

Secretory carcinoma——impact of translocation and gene fusions on salivary gland tumor

Secretory carcinoma（SC）, previously described as mammary analogue secretory carcinoma（MASC）, is a recently described salivary gland tumor which morphologically resembles mammary secretory carcinoma. The first description of SC/MASC, reported by Skálová et al. in 2010, was as a rare salivary carcinoma imitating secretory carcinoma of the breast. SC/MASC is a unique salivary gland tumor with morphological overlap with acinic cell carcinoma（Aci CC）, mucoepidermoid carcinoma（MEC）, and adenocarcinoma not otherwise specified（ADCNOS）. SC/MASC shares similar clinicopathological features with Aci CC. As a critical difference between SC/MASC and Aci CC, SC/MASC characteristically has the chromosomal translocation t（12;15）（p13;q25） which leads to a fusion gene between the ETV6 gene on chromosome 12 and the NTRK3 gene on chromosome 15. This genetic background is an important differential diagnostic finding for excluding other salivary gland tumors and may be a critical factor determining the prognosis for patients with SC/MASC. Research in recent years has provided a large body of new data on SC/MASC and suggests the possibility that the ETV6-NTRK3 translocation could be a therapeutic target. Here, we review the morphological and clinicopathological features of SC/MASC and discuss new directions for therapy.

Construction of the recombinant expression vector for CD80-IgG fusion gene and its expression in Chinese hamster ovary cells

To construct the recombinant expression functionally in Chinese hamster ovary cells in order vector for CD80-IgG fusion gene and to express it to be used as an effective method to eliminate the immune escape of leukemic cells, the cDNA encoding the signal and extracellular domains of murine CD80 was generated by PCR amplification from plasmid pcDNMB7 containing the full length cDNA of murine CD80 and those of murine IgG1, in which the Fc fragment was obtained through RT-PCR amplification from murine spleen cells. These two cDNAs were then cloned in tandem into eukaryotic expression vector pcDNA3.0 and the resultant recombinant plasmid pcDNA/CD80-IgG was then transfected to Chinese hamster ovary cells with liposome transfection reagent. The cell clones constitutively expressing CD80-IgG fusion protein were obtained by G418 screening. Western blotting and dot ELISA assay were used to detect the expression of the fusion protein in the supernatants of these cells. Meanwhile, the fusion protein expressed was then purified with affinity chromatography, and its biological activity was demonstrated by flow cytometry, MTr colorimetry and ELISA assay. The experimental resuits showed that these two inserts were successfully cloned into plasmid pcDNA3.0, and the highly purified fusion protein was obtained. This fusion protein was proved to be able to upregulate the density of CD80 on leukemic cells, deliberately promote the proliferative reactions of mouse allogenic lymphocytes and increase the killing activity against WEHI-3 cells from 49.7 % up to 84.6 %. In addition, this fusion protein could also enhance the IL-2 secretion from allogenic lymphocytes activated by tumorspecific antigens. It is concluded that the recombinant vector constructed can be functionally expressed in the mammalian cells, thus providing a solid foundation for the further investigation on the mechanism to eliminate the immune escape of leukemic cells in vivo.

Paradigm shift of chemotherapy and systemic treatment for biliary tract cancer

Biliary tract cancers(BTC)are frequently identified at late stages and have a poor prognosis due to limited systemic treatment regimens.For more than a decade,the combination of gemcitabine and cis-platin has served as the first-line standard treatment.There are few choices for second-line chemo-therapy.Targeted treatment with fibroblast growth factor receptor 2 inhibitors,neurotrophic tyrosine receptor kinase inhibitors,and isocitrate dehydrogenase 1 inhibitors has had important results.Immune checkpoint inhibitors(ICI)such as pembrolizumab are only used in first-line treatment for microsatellite instability high patients.The TOPAZ-1 trial's outcome is encouraging,and there are several trials underway that might soon put targeted treatment and ICI combos into first-line options.Newer targets and agents for existing goals are being studied,which may represent a paradigm shift in BTC management.Due to a scarcity of targetable mutations and the higher toxicity profile of the current medications,the new category of drugs may occupy a significant role in BTC therapies.