獵頭職場真相觀察

老闆要2個業務聯絡200個客戶...為什麼打完100通電話、「聽話」的人反而被解僱？
<撰文者江湖人稱S姐> 出處商業週刊按我

這是兩位業務在剛開始要做客戶開發時遇到的故事，可以拿來當借鏡。兩位員工暫且稱為小劉和小王好了，而這位主管的管理方式喜歡訓練員工思考。

主管：手邊這200個客戶跟聯繫窗口，你們分別聯繫100個，一周後我們來討論狀態。

小劉：好，我現在立馬來做。

小王 : 這100個名單裡面，哪些是比較重要但被忽略的客戶？哪些是久沒連繫的客戶？

討論一番以後，隔了一周，主管開會時分別和兩位業務詢問客戶狀態。

GRC NPC Index Page | EBV Meeting

• Clevers, Hans
• Delecluse, Henri-Jacques
• Fuchs, Elaine
• Futreal, Andrew
• Hildesheim, Allan
• Lieberman, Paul M.
• Liu, Fei-Fei
• Lo, Kwok-Wei
• Lo, Yuk-Ming Dennis
• Longnecker, Richard
• Lu, Xin
• Ma, Jun | Ngan, Roger | Bei, Jin-Xin
• Rooney, Cliona
• Tsai, Ming-Han
• Tsao, George SH
• Zeng, Mu-Sheng
• zur Hausen, Harald
• Unclassified (Hong Zheng, Honglin Chen, Brigette Ma, Wei Dai | TESSA, TGFBRI inhibitor, BPLF1 DUB)
• Group Photo

GRC NPC - Lieberman, Paul M.

2018 Madison Meeting
HENLE LECTURE
Paul M. Lieberman, Ph.D. The Wistar Institute Philadelphia, PA
Paul Lieberman obtained a Ph.D. at Johns Hopkins University, School of Medicine studying Epstein-Barr Virus reactivation in the laboratory of Dr. Gary and Diane Hayward. He completed a post-doctoral fellowship with Dr. Arnie Berk at UCLA investigating basic mechanisms of transcriptional activation by viral factors. He joined the Roche Institute of Molecular Biology as an Assistant Member and then moved to the Wistar Institute in Philadelphia in 1995. His research focuses on the molecular biology of Epstein-Barr Virus (EBV) and Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) latency and persistence, with particular interests in epigenetic mechanisms and chromosome biology. His work combines biochemical, proteomic, and functional genomic methods to investigate regulation of viral latency, and how this contributes to viral carcinogenesis. The Lieberman group has leveraged this basic research to develop novel small molecule inhibitors and modulators of EBV and KSHV latency to treat viral-associated disease. Lieberman is presently the Hilary Koprowski Endowed Chair and Program Leader in Gene Expression and Regulation and the Director of the Center for Chemical Biology and Translational Medicine at the Wistar Institute.

Arch Pharm Res. 2017 Aug;40(8):894-905. doi: 10.1007/s12272-017-0939-5. Epub 2017 Aug 4.

DNA hypermethylation induced by Epstein-Barr virus in the development of Epstein-Barr virus-associated gastric carcinoma.

Choi SJ1, Shin YS2, Kang BW3, Kim JG3, Won KJ4, Lieberman PM5, Cho H6, Kang H7.

Author information

Abstract

Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is a recently recognized disease entity defined by the presence of EBV in gastric carcinoma cells. EBV infection causes major epigenetic alterations in the EBV genome and its cellular host genome, suggesting that EBV acts as a direct epigenetic driver for EBVaGC. One of the major epigenetic events in the viral and cellular genomes to control transcription is DNA hypo- or hyper-methylation. Particularly, local and global hypermethylation have been reported in EBVaGC. It is therefore important to understand the molecular mechanisms of DNA hypermethylation during EBVaGC carcinogenesis. To understand the functional roles of DNA methylation and suggest therapeutic target candidates for EBVaGC, we reviewed recent literature reporting DNA hypermethylation in EBVaGC. We summarized the identified candidate genes that are markedly hypermethylated in EBVaGC, which can potentially be targets for chemotherapies with demethylating agents. (pmid:28779374)

J Virol. 2017 Aug 9. pii: JVI.00804-17. doi: 10.1128/JVI.00804-17. [Epub ahead of print]

Coordinate Regulation of TET2 and EBNA2 Control DNA Methylation State of Latent Epstein-Barr Virus.

Lu F1, Wiedmer A1, Martin KA1, Wickramasinghe PJMS1, Kossenkov AV1, Lieberman PM2.

Author information

Abstract

Epstein-Barr Virus (EBV) latency and its associated carcinogenesis are regulated by dynamic changes in DNA methylation of both virus and host genomes. We show here that the Ten-Eleven Translocation 2 (TET2) gene, implicated in hydroxymethylation and active DNA demethylation, is a key regulator of EBV latency type DNA methylation patterning. EBV latency types are defined by DNA methylation patterns that restrict expression of viral latency genes. We show that TET2 mRNA and protein expression correlate with the highly demethylated EBV type III latency program permissive for expression of EBNA2, EBNA3s, and LMP transcripts. We show that shRNA depletion of TET2 results in a decrease in latency gene expression, but can also trigger a switch to lytic gene expression. TET2 depletion results in the loss of hydroxymethylated cytosine, and corresponding increase in cytosine methylation at key regulatory regions on the viral and host genomes. This also corresponded to a loss of RBP-jκ binding, and decreased histone H3K4 trimethylation at these sites. Furthermore, we show that the TET2 gene, itself, is regulated similar to the EBV genome. ChIP-Seq revealed that TET2 gene contains EBNA2-dependent RBP-jκ and EBF1 binding sites, and is subject to DNA methylation associated transcriptional silencing similar to EBV latency type III genomes. Finally, we provide evidence that TET2 colocalizes with EBNA2-EBF1-RBP-jκ binding sites, and can interact with EBNA2 by co-immunoprecipitation. Taken together, these findings indicate that TET2 gene transcripts are regulated similarly to EBV type III latency genes, and that TET2 protein is a cofactor of EBNA2 and co-regulator of the EBV type III latency program and DNA methylation state.

IMPORTANCE Epstein-Barr Virus(EBV) latency and carcinogenesis involves the selective epigenetic modification of viral and cellular genes. Here, we show that TET2, a cellular tumor suppressor involved in active DNA demethylation, plays a central role in regulating DNA methylation state during EBV latency. TET2 is coordinately regulated and functionally interacts with the viral oncogene EBNA2. TET2 and EBNA2 function cooperatively to demethylate genes important for EBV-driven B cells growth transformation. (PMID: 28794029)

PLoS Pathog. 2017 Aug 30;13(8):e1006596. doi: 10.1371/journal.ppat.1006596. eCollection 2017 Aug.

Deregulation of KSHV latency conformation by ER-stress and caspase-dependent RAD21-cleavage.

De Leo A1, Chen HS1, Hu CC1, Lieberman PM1.

Author information

Erratum in

• Correction: Deregulation of KSHV latency conformation by ER-stress and caspase-dependent RAD21-cleavage. [PLoS Pathog. 2018]

Abstract

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is a human gammaherpesvirus recognized as the principal causative agent of KS and primary effusion lymphoma (PEL). KSHV establishes persistent latent infection in B lymphocytes where viral gene expression is restricted, in part, by a cohesin-dependent chromosome conformation. Here, we show that endoplasmic reticulum (ER) stress induces a rapid, caspase-dependent cleavage of cohesin subunit RAD21. ER stress-induced cleavage of RAD21 correlated with a rapid and strong viral lytic transcriptional activation. This effect was observed in several KSHV positive PEL cells, but not in other B-cells or non-B-cell models of KSHV latency. The cleaved-RAD21 does not dissociate from viral genomes, nor disassemble from other components of the cohesin complex. However, RAD21 cleavage correlated with the disruption of the latency genome conformation as revealed by chromosome conformation capture (3C). Ectopic expression of C-terminal RAD21 cleaved form could partially induce KSHV lytic genes transcription in BCBLI cells, suggesting that ER-stress induced RAD21 cleavage was sufficient to induce KSHV reactivation from latency in PEL cells. Taken together our results reveal a novel aspect for control and maintenance of KSHV genome latency conformation mediated by stress-induced RAD21 cleavage. Our studies also suggest that RAD21 cleavage may be a general regulatory mechanism for rapid alteration of cellular chromosome conformation and cohesin-dependent transcription regulation. (PubMed: 28854249)

PLoS Pathog. 2018 Jan 19;14(1):e1006844. doi: 10.1371/journal.ppat.1006844. eCollection 2018 Jan.

RNA-Seq of Kaposi's sarcoma reveals alterations in glucose and lipid metabolism.

Tso FY1, Kossenkov AV2, Lidenge SJ1,3,4, Ngalamika O5, Ngowi JR3, Mwaiselage J3,4, Wickramasinghe J2, Kwon EH1, West JT1, Lieberman PM2, Wood C1.

Author information

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS). It is endemic in a number of sub-Saharan African countries with infection rate of >50%. The high prevalence of HIV-1 coupled with late presentation of advanced cancer staging make KS the leading cancer in the region with poor prognosis and high mortality. Disease markers and cellular functions associated with KS tumorigenesis remain ill-defined. Several studies have attempted to investigate changes of the gene profile with in vitro infection of monoculture models, which are not likely to reflect the cellular complexity of the in vivo lesion environment. Our approach is to characterize and compare the gene expression profile in KS lesions versus non-cancer tissues from the same individual. Such comparisons could identify pathways critical for KS formation and maintenance. This is the first study that utilized high throughput RNA-seq to characterize the viral and cellular transcriptome in tumor and non-cancer biopsies of African epidemic KS patients. These patients were treated anti-retroviral therapy with undetectable HIV-1 plasma viral load. We found remarkable variability in the viral transcriptome among these patients, with viral latency and immune modulation genes most abundantly expressed. The presence of KSHV also significantly affected the cellular transcriptome profile. Specifically, genes involved in lipid and glucose metabolism disorder pathways were substantially affected. Moreover, infiltration of immune cells into the tumor did not prevent KS formation, suggesting some functional deficits of these cells. Lastly, we found only minimal overlaps between our in vivo cellular transcriptome dataset with those from in vitro studies, reflecting the limitation of in vitro models in representing tumor lesions. These findings could lead to the identification of diagnostic and therapeutic markers for KS, and will provide bases for further mechanistic studies on the functions of both viral and cellular genes that are involved. (PubMed: 29352292)

2016 GRC NPC

GRC NPC Group Photo_2016

GRC NPC - Tsai, Ming-Han

2018 GRC NPC

• 1 ng/ml IL8 for 7 days —> Zta expression
• Transcriptome data
• Small RNA-associated TLRs 
• TLR3, TLR7 
• TLR7  is involved..

2016 GRC NPC

GRC NPC - Rooney, Cliona

Cliona Rooney (Baylor College of Medicine, USA)
"Cancer Immunotherapy and Challenges for NPC"

1. T cell for each patient, pre existing , 22% chance response
2. Vaccine an Ad-based LMP2, MVA-EBNA1-LMP2
3. LCL activated vEBVST
4. Singpore TESSA therapeutics (http://goo.gl/zdbYpA) 330 patients (與Wed Poster #5相關)
5. Ad LMP1/2/LCL activated T cell for NPC, not better for EBVST, but is better in lymphoma patients (type 2 malignancy lymphoma)
6. SFC, IFNG coated plate, 6 month 1.5 year
7. survivors epitome spread: MAGEA4 + SURVIVIN + PRAME
8. Dendritic cells are difficult to make in NPC patients
9. 1st stimulation: EBV EBNA1 ,LMP1, LMP2, BARF1
10. 2nd stimulation: Pepmix all possible MHC2
11. GRACE trial (patients of lymphoma)
12. Switch to IL15/7
13. High dose IL15 increase specificity
14. Fewer CD4 cells in high dose IL15 (SF: IL21 and IL15 are part of the gamma-chain cytokine family and are crucial for survival and proliferation of Tfh (follicular Th), cytotoxic and memory T cells, both IL21 and IL15 are being used in clinical trial)
15. IL15H increases central key EBVSTs
16. Post infusion expansion of EBVSTs in patients with lymphoma
17. heterogeneity in NPC TME
18. Importance for epitope spreading, maybe viral lytic genes are important (Mei-Ru asked what kind of lytic genes in her mind, she replied: IE genes)
19. TGFBR
20. Infiltrating barrier
21. Gottschalk PI is responsible
22. Lympho depletion/Reactivate EBV
23. Immuno checkpoint
24. Genetic TGFBR DN to increase T cells
25. Steve Rosenberg was mentioned again and again and again (https://goo.gl/LKXqYG)
26. T cells express NPC expressing cytokine 2 receptor to "homing"
27. GuangZhou published some promising results (ref: Oncoimmunology 2015)

From Ming-Han 

1. 1 Small molecules? (one drug for all patients)
2. 2 EBV-specific T cells;
3. 3 Chimeric antigen receptor (CAR) T cells?
4. 4 Gene-modified T cells? (EBVSTs)
5. One patient one drugs?

Ongoing trials

1. Chiun Hsu (許駿); NTU PD1 inhibitor (KEYTZUDA KEYNOTE 028 Overall Response rate 10.8 %)
2. LMP2 peptide pulsed DCs (HK): increase LMP2A specific T cells
3. Ad-dLMP1-LMP2 transduced DCs (Singapore by Prof. Toh): DTH but no elevated LMP specific T cells.
4. MVA-EBNA1-LMP2: functional in 50% patients with the elevated T cell responses.
5. LCL-activated EBVSTs for NPC (adjuvant (62%) remain diseases free / 2nd or relapse in 48.7%)
6. LCL activated EBVSTs after Chemotherapy? (Singapore (Toh, Ho): 35 patients; 97% type III diseases; Gemicitabin and carboplatin 4 cycles. (combine treatment?); 71.4% responses; overall survival 2yr 65%; 5yr 35%.)
7. Gemicibin alone vs Gemicibin + EBVSTs on going in 350 patients. (Ongoing)

Ad5f35-∆LMP1-LMP2 (Adenovim)

• infect PBMC --> boosted by EBV-LCLs (in between plus IL2) ---> wish to have LMP2A/1 T cell CTLs. (Bollard et al, J, Clin Onco 2014 32 (8) 798-808.)

EBVST treat for EBV lymphoma (CTC project)

to NKT lymphoma (remission 25/26)
- Epitope spreading after treatment of EBVSTs seems very important.

This strategy seems good BUT problems

• too long
• No LCL available from these EBV+lymphoma patients; either low B cell or the B cells can't be immortalised by EBV for?? reason.
• Require live virus.
• Many cases all T cells only in the end recognise EBNA3C.

Original plan:

• (PBMC infected by B95-8) --> LCL --> stimulated with DCs with LMP1/2A ---> T cells (peptide of LMP2A by using many many peptides; each 15mer but with 11aa overlapping each)

Now: 

• PBMC+peptide --> (+IL4/7) can have expansion 100X times T cells only within 10 days; enough time to treat PTLD.

Issue:

• This strategy works super well in healthy donor BUT IL4/7 doesn't work well for expansion of patient's T cell for ?? reason.

Phase I clinical trial design (GRACE)

• HL, BL… EBV+ lymphoma.
• Patients T cells with very low avidity;
• Patient with lymphoma have very few T cells.
• Growth of T cells from patients always badly.

New strategy:

IL4/7 vs IL7/15? which is better: 
found IL7/15 is better (high dose IL15: 100ng/ml; medium dose IL15: ? ng/ml)
But still IL7/15 still difficult to expand T cells from patients.

             IL4+IL7       IL7+IL15     IL7+IL15hi
CD4          50%          30%                ~0
CD8          50%          70%             ~100 %

Problem2: LMP1/2 actually not expressed in all tumor cells. For example; if stimulate T cells with BARF1, LMP2, EBNA1 or LMP1, in the end T cells only recognise one peptides…

Epitope spreading: 

• SSX2, NYESP, Survivin, Prame, Mage4 ---> these antigens only exists in lymphoma.
• after treatment patients have T cells against these peptides then the treatments outcome will be better.
• So far: 2/10 cases total cure; 4/10 with better response.

For NPC cases: 

• EBVSTs have less responses for NPC patients
• Possible reasons:  solid tumor; difficult for T cells to enter; Immune suppressive environment.

Requirements if want to treat NPC:

• migration to tumor site
• Infiltrate tumor parenchtmas?
• have to proliferate at tumor sites that express also MHCI

@Gottschalk (NPC clinical trials)
@Effective CAR and TIL stimulation
Requirement for human trials: GMP director; CMP CTL; everything GMP

-------------------------------- Q1: CD4/8 mix during T cell expansion; which one is better?

A1: hard to say. Some successfully with 90% CD4 the others with 90% CD8 but she thinks 50% 50% is the best.

Q2: Epitope spreading: spreading to cancer-associating T cells with viral antigens?
A2: not viral antigens.

Q3: LCL or peptide to stimulate T cells? why difference?
A3: use bacteria or baculovirus to stimulate? Different method might lose the chance to have correct epitopes; for example, phosphorylation. Now we don't use LCL to stimulate T cells; we use adenovirus-infected DCs it's better.

Q5: someone firstly transgene the T cells with specific chemokine receptor corresponding to chemokines secreted by tumor cells; is that good? Yes.

Q6: Combine EBVSTs with chemotherapy? which first?
A6: Suggest to firstly to irradiation or chemotherapy then inject T cells since tumor necrosis can attract T cells to the tumor sites.

In EBV Meeting Invited lecture on immunology, Plenary lecture hall KOH-B-10
Chairperson: Cliona Rooney, Baylor College of Medicine, Houston, USA
Lecturer: Jeffrey Cohen, National Institutes of Allergy and Infectious Diseases

GRC NPC - Zeng, Mu-Sheng

2018 GRC NPC

Mu Sheng Zeng (Sun Yat-Sen University Cancer Center, China)
"The Key Cellular Factors in EBV Infection of Epithelial Cells"

• 4912 new cases NPC in 2016
• Bmi-1 induce immortalization of NOC cells
• NPEC1-BMI-1 And NPEC2-BMI-1. Has 10 fold higher efficiency for EBV infection (cell free)
• NMHC-IIA is a poteintial key factor in EBV infection
• NMHC-IIA interacted with gHgL and co-local
• Membrane redistribution f NMHC-IIA which localized with gHgL

• EGF unregulated 5 membrane proteins. (AREG/NT5E, EPHA2, F3, EGFL5, DCBCL2)
• KO down of EPHA2 markedly decreased EBV infection
• gH/gL and gB interact with EPHA2
• NRP1 — gB CendR domain
• NRP1 is a receptor of HTLV
• EBV entered epithelial cells via lipid raft
• EBV internalizes into eputhelial cells in the presence of NRP1 and EphA1
• EBV fuses with endosomatic membrane by binding to EphA2

• Genetic susceptibility of NPC patients
• Genetic variation of EBV strains (full-length sequences of EBV
• Epithelial cell receptors for EBV infection
• Highly susceptible NPCx model
• Cellular factors (The mechanism of EBV infection and transformation in nasopharyngeal carcinoma)

2016 GRC NPC

Mu-Sheng Zeng (Sun Yat-Sen University Cancer Center, China)"NPC, an Epithelial Cancer: Immortalization and Tumorigenesis"  曾木聖

1. NMHC-IIA and NRP1 mediate EBV infection by interaction with gHgL and GB，respectively
2. NRP1 mediated EGFR signaling is involved in EBV infection
3. NP69 + Bmi1 + CCND1 enhances EBV akata EGFP infection to 30%
4. Sphere like
5. FGFR3-TACC3 (low prevalence in NPC after big-screening)
6. ncRNA: RBM24 suppresses CNE2 growth
7. MALAT1, miR-25 inhibits MALAT1 expression
8. 中山大學Nature子刊揭示病毒致癌新機制 (http://goo.gl/dUieQt)
9. Cell子刊：首張致癌EB病毒註釋圖譜 (http://goo.gl/3YsJIs)

From Ming-Han

1. LMP2A cause EMT?
2. receptor of epithelial cells?
3. How EBV infects epithelial cells? hints (1) very low infectivity; (2) usually clonal EBV. (Dougles Hanahan Cell 2011)

Establish immortalised NPECs

1. SV40LT
2. HPV E6 E7
3. Bim-1 (GOOD!) and also highly expressed in bad NPC cases.

How? 

1. Overexpress Bmi1 immortalised NP cells by reduce p16; 
2. over express hTERT and stemness pathway.

Now in their lab:

1. Bmi1 + hTert or Bmi1 + p16ko: can easily immortalise NP cells and after EBV infection EBV can easily keep inside the cells and keep in latency II.
2. Whether EBV infection cause transform and tumorigenesis?

• -in cell infection model (2015)?
• -gp110->CRGDK/R->Neuropilin-1 (NRP-1+gb) increase of NP cells (2015 nature communications)
• -infection of EBV mainly mediated by macropinocytosis; together with EBV bound to NRP-1

Question: 
EBV gene expression in NPC by pair-ended transcriptomes
(1) some type III latency may be express in primary NPC tissues and C666 cell line (Fu LJ, teal Front med 2016)

How about cellular gene? any fusion genes in NPC? since it will be good target for drugs.
FGFR3-TACC3 fusion gene: 5% of NPC. (SF: 這個沒搞頭)
RBM24 and NPC: miR25 is down in NPC; over expression of miR25 Block NPC grow and invasion

Q: target of miR25?
@@MALAT1: highly possible target.

model: RBM24hi-> more mir@5-> block MALAT1-> no tumor or vice versa.

Conclusion:

• 1 Bmi1, htert and kd of p16 important of NPEC
• 2 Bmi-1 and cyclinD1 support EBV latent infection in NPECs.
• 3 Sphere-like culture of Bmi-1 immortalised cells support EBV infection
• 4 NHMC-IIA and NRP-1 mediate EBV infection. Importantly; these proteins expressed at low level of normal NPECs but highly in NPCs.   http://www.ncbi.nlm.nih.gov/nuccore/323276615

In August EBV Meeting Session 11: EBV and Autoimmunity
Cellular Factors in Epstein-Barr Virus Infection of Nasopharyngeal Epithelial Cells
Mu-Sheng Zeng, Sun Yat-Sen University Cancer Center, Guangzhou, China

GRC NPC - Delecluse, HJ

Henri-Jacques Delecluse (DKFZ - German Cancer Research Center, Germany) "Is NPC Caused by a Particular EBV?"

1. Kowk et al 2014 J Virol phylogenetic tree
2. Palsler et al JV 2015 EBV polymorphism
3. M81 and B95-8 difference Tsai et al Cell Reports 2013 ((Tsai et al., Cell Rep, 2013 Spontaneous lytic replication and epitheliotropism define an Epstein-Barr virus strain found in carcinomas)
4. J Mautner will be reporting in Switzerland regarding epitope polymorphism M81 B958 AG (B95-8 and M81: CD4 epitope only 30% conserved; CD8 only 70% conserved.)
5. The mucosa that covers sinuses is a good source of respiratory
6. Transfer infection vs direct infection
7. Involcurin is a marker for late differentiation
8. M81 and B95-8 express gp110 at different levels
9. The Epstein-Barr Virus BART miRNA Cluster of the M81 Strain Modulates Multiple Functions in Primary B Cells ((Lin et al., PLoS Pathog, 2015 The Epstein-Barr Virus BART miRNA Cluster of the M81 Strain Modulates Multiple Functions in Primary B Cells)
10. M81 replicates in vivo
11. Functions served by the BART miRNAs
12. BART miRNAs inhibit BZLF1 expression
13. The BART miRNA also inhibits LMP1 expression
14. Deletion of the BART miRNAs increases infectivity in NSG mice
15. BART inhibit LMP1 expression
16. Pro-apoptotic (caspase 3) vs anti-apoptotic (LMP1)

-------------------------------------------- (以下未發表)

1. Polymorphism issue
2. 14 EBVL, 9 IM, GP202 Korean gastric cancer, 30 days - 40 days all NSG mice died. Except some.
3. Replicating viruses are more tumorigenic in vivo
4. Aneuploidy after M81 infection: M81 WT > delta ZR
5. Cliona Rooney commented: John Sixby published BCLF1 increases aneuploidy

From Ming-Han
From HJD news:
B95-8 and M81: CD4 epitope only 30% conserved; CD8 only 70% conserved. 
Sphenoidal sinus (Respiration sinus)

Question: BART and epi cells? ongoing
Q: try to study polyorphisms? Yes
Q: by Paul Lieberman  why M81 more lytic but in Asia lymphoma is low? 
A: the balance between grow rate and lytic replication?

In EBV Meeting
Session 2: B Cell Infection
The Epstein-Barr virus BART miRNA cluster of the nasopharyngeal carcinoma- associated M81 strain modulates lytic replication and cell growth in primary B cells
Ming-Han Tsai, German Cancer Research Institute, Heidelberg, Germany

Session 13: Adaptive Immunity to EBV
Epstein-Barr virus strain heterogeneity impairs T cell immunity
Josef Mautner, Helmholtz Center Munich, Germany

GRC NPC - Tsao, George SH

George Tsao (University of Hong Kong, Hong Kong SAR China)"Establishment of New NPC Xenografts and Cell Lines for EBV Studies"  曹世華

1. Int J Cancer Vol 83 1999 Lim (C-6661)
2. elaborate on NPC-HeLa hybrid contamination available NPC cell lines (refers to KW Lo’s paper and JV Strong RNA-seq paper)
3. Surgical Maxi William
4. NPC Xeno43 65M recurrent NPC: The first xenograft line established from clinical tissue (TI，T2，T3，T4，T5，T6，T7 then … no longer passable) (inoculated on the top of mouse kidney)
5. NPC Xeno76 stage1，primary NPC (1. EM examination of EBV virus 2 . WGS of Xenograft and cell line )
6. For cell line establishment, Tsao suggested the use of 4 μM Y27632, a Rho kinase inhibitor) based on (Liu et al., Am J Pathol, 2012)
7. Rapid drop down of EBV EBER copies
8. NPC Xeno47, 52F recurrent, take out of FCS，replenish with GF (avoid differentiation)
9. C17 spheroids RPMI with 10% FBS + 4 μM Y27632
10. NPC38: a EBV negative NPC cell line
11. RNAseq
12. NRAS Q61R Q61K 2/4 ( think about drugs。)
13. TP53 mutation G245D
14. KMT2C/KMT2D
15. CYLD mutation and BCL3 and Deubiqnitization
16. Notes: 徐正龍醫師的NPC Xeno則多為 meta, 他在Oncotarget的paper (Hsu et al., Oncotarget, 2015)

From Ming-Han

• Issue: need to keep these precious samples.
• Method: Xenograft into Nude mouse kidney capsules.
• Problems: (1) loss EBV quickly; (2) stop growing

Previous Results and issues:

• in 20% cases; HPV18 positive!! (Chen et al IJC 1998)
• Before 2008; nearly all NPC cell lines are E7 or E6 HPV positive!!?
• Decide to make new ones in case doing something wrong studies.

Surgery:

• Cut a small hole of nude mice from the back.
• Squeeze the kidney out
• implant 3 small NPC primary tumours
• put back kidney and wait.

Result:

• they found the tumor grows faster and faster if they re-implant the "grow-up" tumor back to new nude mice.
• Successfully rate: (2-3/40 (T2); 2-3/17 (T3))
• For the ones failed to grow up: 1. central parts of tumor are necrosis; 2. highly in lytic replication.

American Journal of Pathology vol 180, N2, 2012 

ROCK inhibitor (27632) can make epithelial cells grow indefinitely no matter with tumor or without tumor. (4 uM is the best!!) can grow 1000 days. if using ROCK inhibitor to cultivate NPC cell lines?

1. Yes, can grow
2. Chromosome become a mass
3. 95% EBV+ cell after culture
4. P1-P10 (100 EBV copy/cell); >P32 (drop to 10 copy EBV/cell)
5. These cells can be injected back to mice
6. The removal of Y27632: cell stop growing?? and turn into lytic!!

Further

1. PD80 cell line (very old) if plus TPA+SB: still lytic!! can collect virus and infect AkataEBV- cell and induced virus by anti-IgG
2. Final have 1 primary cell line NPC47C; around PD90, 100% EBV+.

---------------------------------------------------------------------------

Question: try spheroids cell culture? Can they grow into 3D?

Ans: Yes, but grow very slowly.

Finally, sequence the tumor genome before or after Xenografts.

NRAS mutations in NPC? (2/4 in Xenograft, 2/40 in primary NPC)

TP53 mutation higher.

KMT2D, CYLD, TRAF3, P53 higher mutated in Xenograft

Q1: what's CYLD's function? Deubiquitin?

A1: Can inhibit NFKB activation and may change the localisation of BCL3 from the nucleus to cytosol. BCL3 also involved in NFKB activation.

Q2: EBV-NPC: only isolated from recurrent NPC; what's the genomic hallmarks inside?

A2: Huge Huge amount of genomic alteration compared to the fresh-isolated tumours.

Q3: EBV can block ATM pathway

Q4: NPC interestingly can easily recurrent back after 5 years or even longer but H and N tumor usually not. (這點和和信鄭主任所述相同)

Q5: Need to take care whether these Xenograft cell lines be contaminated by murine viruses. (SF: Oh my god!)

EBV Meeting Session 10: Nasopharyngeal carcinoma and other epithelial cell tumors

"Establishment and characterization of newly established nasopharyngeal carcinoma xenografts and in vitro NPC cell lines for EBV studies"

George Tsao, University of Hong Kong, China

GRC NPC - Ma, J and Bei, JX

2018 GRC NPC

Jun Ma (Sun Yat-Sen Medical University, China)
"Comprehensive Treatment Strategy in Locoregionally Advanced Nasopharyngeal Carcinoma"

• Evolving National Comprehensive Cancer Network's (NCCN) guidelines (2018) of the nasopharynx

Lancet Oncol. 2016 Nov;17(11):1509-1520. 

Induction chemotherapy plus concurrent chemoradiotherapy versus concurrent chemoradiotherapy alone in locoregionally advanced nasopharyngeal carcinoma: a phase 3, multicentre, randomised controlled trial.

Sun Y1, Li WF1, Chen NY2, Zhang N3, Hu GQ4, Xie FY1, Sun Y5, Chen XZ6, Li JG7, Zhu XD8, Hu CS9, Xu XY10, Chen YY6, Hu WH1, Guo L11, Mo HY11, Chen L1, Mao YP1, Sun R1, Ai P2, Liang SB3, Long GX4, Zheng BM5, Feng XL6, Gong XC7, Li L8, Shen CY9, Xu JY10, Guo Y12, Chen YM13, Zhang F1, Lin L1, Tang LL1, Liu MZ1, Ma J14.

Abstract

BACKGROUND:

The value of adding cisplatin, fluorouracil, and docetaxel (TPF) induction chemotherapy to concurrent chemoradiotherapy in locoregionally advanced nasopharyngeal carcinoma is unclear. We aimed to compare TPF induction chemotherapy plus concurrent chemoradiotherapy with concurrent chemoradiotherapy alone in a suitably powered trial.

METHODS:

We did an open-label, phase 3, multicentre, randomised controlled trial at ten institutions in China. Patients with previously untreated, stage III-IVB (except T3-4N0) nasopharyngeal carcinoma, aged 18-59 years without severe comorbidities were enrolled. Eligible patients were randomly assigned (1:1) to receive induction chemotherapy plus concurrent chemoradiotherapy or concurrent chemoradiotherapy alone (three cycles of 100 mg/m2 cisplatin every 3 weeks, concurrently with intensity-modulated radiotherapy). 

Lancet Oncol. 2018 Mar;19(3):382-393. 

Development and validation of a gene expression-based signature to predict distant metastasis in locoregionally advanced nasopharyngeal carcinoma: a retrospective, multicentre, cohort study.

Tang XR1, Li YQ1, Liang SB2, Jiang W3, Liu F4, Ge WX5, Tang LL1, Mao YP1, He QM1, Yang XJ1, Zhang Y1, Wen X1, Zhang J1, Wang YQ1, Zhang PP1, Sun Y1, Yun JP1, Zeng J1, Li L1, Liu LZ1, Liu N1, Ma J6.

BACKGROUND:

Gene expression patterns can be used as prognostic biomarkers in various types of cancers. We aimed to identify a gene expression pattern for individual distant metastatic risk assessment in patients with locoregionally advanced nasopharyngeal carcinoma.

METHODS:

In this multicentre, retrospective, cohort analysis, we included 937 patients with locoregionally advanced nasopharyngeal carcinoma from three Chinese hospitals: the Sun Yat-sen University Cancer Center (Guangzhou, China), the Affiliated Hospital of Guilin Medical University (Guilin, China), and the First People's Hospital of Foshan (Foshan, China). Using microarray analysis, we profiled mRNA gene expression between 24 paired locoregionally advanced nasopharyngeal carcinoma tumours from patients at Sun Yat-sen University Cancer Center with or without distant metastasis after radical treatment. Differentially expressed genes were examined using digital expression profiling in a training cohort (Guangzhou training cohort; n=410) to build a gene classifier using a penalised regression model. We validated the prognostic accuracy of this gene classifier in an internal validation cohort (Guangzhou internal validation cohort, n=204) and two external independent cohorts (Guilin cohort, n=165; Foshan cohort, n=158). The primary endpoint was distant metastasis-free survival. Secondary endpoints were disease-free survival and overall survival.

對啊，那是我發的!!!

2016 GRC NPC

Jun Ma 馬駿 (Sun Yat-Sen Medical University, China) 
"NPC Clinical Trials in Guangzhou"

Roger Ngan (Queen Elizabeth Hospital, Hong Kong SAR China) 
"Evolution of NPC Clinical Trials in Hong Kong"

中山大學附屬腫瘤醫院馬駿團隊的研究成果寫入美國教科書成美歐指南 (http://goo.gl/mPcswZ)

1. 3500 NPC patient /yr (In their institute)
2. 12台 Linear particle accelerator (LINAC) treating 800 patients /day
3. Landmar 0099 trial in 1988 (J Clin Oncol 1998 and Lancet Oncol 2012)
4. NCT    submitted
5. NCT01872968 recruiting
6. NCT3211476 13 centers
7. Radiotherapy is for early stage patients (T1-->2N1  RT or RT+CCRT)
8. advanced : T3+T4
9. IMRT + CCRT did not improve survival !! (因為IMRT是依image打雷射，通常處理地很好, 加了CCRt反倒讓病人增加許多chemotherapy之副作用) 

Jin-Xin Bei (Sun Yat-Sen University Cancer Center, China) 

"Genetic Basis of Nasopharyngeal Carcinoma" 貝錦新

1. Bai-Yue origin (Wee et al., 2010)
2. HLA linkage (HLA-A2 Simons et al 1974, HLA locus Lu et al 1990)
3. Ch 4.2, Ch 4.177, Ch2.1 (Feng et al, 2002, Xiong et al, 2004, Hu et al 2008, Hildeshiam et al 2012, Bai et al, 2012)
4. Candidate gene approach )awaits for validation)
5. NPC GWAS (ch3 MECOM and ITAG9, ch13 TRFRSF1, ch6 HLA-A x 2)
6. However, these GWAS studies only exprealined 2.2% genetic variance (HLA II and CIITA (controls EBV lytic replication,  Manolio et al Nature 2009)
7. Extended GWAS studies (Cui et al., Hum Mol Gene accepted)
8. Exome-wide association study (KLHDC4) Lian et al PLoS One 2016
9. The best predictive model so far: EBV variants (including CIITA and 7 host SNPs Cui et al, Oncotarget, accepted )
10. EBV subtypes (Feng et al., Chin J Cancer)
11. NKT cell lymphoma DPB1-aa 84 (Lancet Oncol)

瑪麗亞，別忘了廣西呀

--------------------------

From Ming-Han

1. @Whether NPC high incidence population all migrated from "Bai-Yun" village of China? (Wee et al 2010)
2. -EBV subtype to NPC? General population+environmental factor cause NPC? Genetic (HLA-A2) cause NPC?

Three highly mutated regions specific to NPC:

1. TNFRSF19
2. MECOM
3. ITAG9

Cui et al: human Mol Genetics (accepts): study >1000NPC and control genetics.

GRC NPC - Longnecker, R

Richard Longnecker (Northwestern University, USA)
"EBV Entry into Host"

Lindsey Hutt-Fletcher retires last year. Give all the reagents to Longnecker.
Cell-cell fusion
gp42 and MHC II structure.
Soluble gb42 stimulates fusion with B cells but inhibits fusion with epithelials.

From Ming-Han
Longnecker: EBV entry
gp350 gHgL gB BMRF2
gB( gp110) is highly conserved in Herpesvirus
gHgL against avb6)
gHgL+gp42: against MHCII   3D structure
(Mbio 2012)
KGD motif, blocking ab to gHgL also block fusion of eli cells (E1D1, CL40, CL59)
gB of EBV is more hydrophobic at C terminal domain

Conclusion:
gHgLgp42: B cell

gHgL: epi cells

soluble gp42: inhibit infection of B cells

Important domain of gHgL by protein striation.

Q: try to pull down receptor? A: try to do IP/Mass but nothing.

Q: B/epi cell infection? A: B cell use endocytosis as vesicle; epi cell firstly need to fused with plasma membrane then enter.

Q: virus entry cytosol; how many entry nucleus?

Q: integrin KO-> EBV infection can elevate or not; A: depend on cell type.

GRC NPC - Lo, Yuk-Ming Dennis

2016 GRC NPC

Dennis Lo (Chinese University of Hong Kong, Hong Kong SAR China) 
"Plasma DNA Analysis for Detecting NPC: Biological Insights and Clinical Applications"
盧煜明 (Wiki https://goo.gl/HTNGFn)
香港中文大學網站 https://goo.gl/03EPTe    http://goo.gl/i7PNRf )

1. Size based molecular diagnosis (difference between maternal and fetal , because the fetal DNA has no linker, therefore is 10 bp shorter) (Lo et al., Lancet, 2003; Chiu et al., BMJ, 2011)
2. Liquid biopsy, HCV 112 E1317 , Ch 1 or 8, amplified vs deleted (Jiang et al., Proc Natl Acad Sci U S A, 2015)
3. Plasma DNA tissue mapping, Deduction of tissue contribution
4. DNA mixtures  methylation DNA (Sun et al., Proc Natl Acad Sci U S A, 2015) The Epigenome Road Map was mentioned
5. Noninvasive prenatal testing (NIPT) (Jiang et al., Trends Genet, 2016; Wong et al., Annu Rev Med, 2016)
6. Characteristics  of plasma EBV DNA: < 181 bp, Short half life, prospective study 201,121, 5% positive for EBV DNA -> 4 wks later, 1.255
7. 30 millions epigenetic DNA markers commercial available