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

GRC NPC - Fuchs, Elaine

Elaine Fuchs (The Rockefeller University, USA HHMI Investigator) 
"Transition of Normal Epithelia to Cancer"
Webpage http://www.rockefeller.edu/research/faculty/labheads/ElaineFuchs/#content

• a student of Howard Green, who just passed away several mo ago
• The foundation of embryonic stem cell culture to come in 1980s burn therapy
• quiescent stem cell and primed stem cells awaiting for activation
• Hair follicle stem cells
• BMP signaling governs stem cell quiescence
• Lossof Foxc1 abrogated stem cell quiescence
• Stem cell specific bind to 400 large open chromatin domains called super-enhancer
• SOX 9, BMP
• Epicenters posses the information needed for a gene to get to a niche microenvironment
• in vitro, HF stem cell transcription factors are downregulated by SOX 9 (ref: Adams and Yang and Fuchs)
• SCC cancers
• Tumor initiating SCC stem cells
• Super-enhancer SCC  (eLife 2015  Yang ETS family transcriptional regulators drive chromatin dynamics and malignancy in squamous cell carcinomas.)
• Activated ETS2 (ref: Oshimori and Fuchs Cell 2015 TGF-β promotes heterogeneity and drug resistance in squamous cell carcinoma)
• TGFb responsiveness in primary tumors
• HRAS v12
• Sox9 is shared in both key HF SC genes and key SCC SC genes
• ETS2 or ELK3 (ref: Foulds et al)
• Genetic lineage tracing (developed by Hans Clever Group Identification of stem cells in small intestine and colon by marker gene Lgr5.)
• Some cells have TGFBR receive TGFb
• TME heterogeneity determine stem cell variations (TGF-β Promotes Heterogeneity and Drug Resistance in Squamous Cell Carcinoma)

---------------------------------------------------------------------------------
From Ming-Han

• Main model: hair cells
• Main question: (1) how grow; how not grow; why go into white hair?
• find stem cells (fast grow (TGFB-); grow fast but not increase. (slow (TGFB+); grow slow near vessel)
• Use lineage tracing techniques to track the fate of the cells
• Strategy
• Use lentivirus to infect d5 embryo: reason: at this stage the skin layer is monolayer in embryo.
• TGF-b responding stem cells have resistant to Cisplatin.
• For epithelial cells
• BSA+cisplatin: all dead
• TGFb+Cisplatin: not death; become flatten, can use for drug screening? (TGFb is important for microenvironment and vessel formation)
• @@Read : lineage tracing techniques!! Can trace the fate of the cells!! Shimori  and Fuches Cell 2015

GRC NPC - Clevers, H

Hans Clevers (Hubrecht Institute, The Netherlands)
"Wnt Signaling and Cancer"

• The champion of all stem cells : crypt base paneth cells
• Wnt is required to maintain crypt stem cells in colon cancer，Wnt is locked in On state。
• Lqr5 is an unusual component that marks stem cells in the crypts。
• 15 stem cell to begin  (mixed with1 Lqr5)  
• Re-tracing 
• Not asymmetric
• Extensive plasticity in crypts
• Lgr5-driven GFP in crypt base columnar cells
• Intestinal stem cells are not quiescent/not rare ~ 18%/not asymmetric
• Lgr5 +ve stem cells are the origin of colon cancer stem cells
• Adenomas retain Paneth stem cell
• Deleting floxed 
• A single  lgr5 cell in a red sermons behaves linebackers stem cells
• Lgr5 uses the receptor : R-spondin
• E3 ligase ZNRF3/RNF43
• R spondin binds Lgr5 and ZNRF3 that stabilize WNT complex
• Single LGR5 stem cell grown in vitro
• 1. R SPONDIN LGR5 2. EGF  3. Noggin (TGFB/ BMP) inhibitor
• Self renewal in stomach corpus glands Troy and Lgr5
• Human gastric organoid and Helicobacter 
• Human Lung organoid
• chemotaxis toward RSV infected lung organoid 
• A living cancer bank with SU2C
• Lineage-tracing: Nat 2007 Identification of stem cells in small intestine and colon by marker gene Lgr5.

From Ming-Han

Wnt signal: crosstalk between cells and shape the crypts of stem cells in gut.

• -Lgr5 and Wnt signal.
• -Wnt block B-cat be degraded by APC-E3 complex; that make tx factor TCF be activated. Once Wnt is weaken, the B-catenin can be releases from APC complex and go to nucleus.
• @Alkaline phosphatase: marks entrocytes lineages. (not mark stem cells!!)
• Lgr5+ cells re stem cells. (Lgr5CRE+ cells: can marked stem cells).
• Lgr5+ cells + R-spondin1 (Wnt Angonist) + EGF + Noggin (TGF inhibitor) + Matrigel ----> can form 3D crypts!! mini gut.
• --> Can inject these mini gut bact to mice which contain the damage of gut--> these small guts can cover the damaged place and fix the damage!!
• -> They expand the studies into

Gastric system:

• Stomach: gastric epithelial cell replace much slower than Gut. The stem cells called "chief cells from isthmus".
• Only Troy+ cells yield stomach organdies and can be infected by Helicobacteria!!
• They apply Casp9 system to modify the gene in organdies.

Lung Organoids:

• Can produce Mucus! and can be infected by pathogens; for example: RSV

Urine:

• p63+ from bladder and Pax8+ from Urine.

Application for their study:

• Virology/Immunology
• Drug screening
• Tumor stem cells/ genomic screening.
• They found that the organdies made by cancer cells look totally different from the normal cells!! Can use single cell culture to sort them out and trace back to "tumor stem cells".
• Apply Caspase9 system to study the genomic and the formation of organdies.

Special and attracting techniques: Lineage-tracing techniques (Identification of stem cells in small intestine and colon by marker gene Lgr5). (read)

Special experiment:

• They use Casp9 system to KO P53 or p16 or SMAD or….
• They found the organoids with these KO -> grow much slower than the normal cells.
• Certain KO don't require EGF anymore.
• After injecting back to mice; although these KO organoids grow badly in vitro but can form tumours in vivo. The healthy ones can't.

GRC NPC - Lo, KW

2018 GRC NPC

• [NPC tumorigenesis model]
• WES-seq of 111 NPC patient Nat Commun 18: 14121, 2017
• LCM enrichment of tumor cells. -> higher somatic mutation than the Singapore study (because of LCM?)
• LMP1 activates both canonical and non-canonical NFKB pathways
• Homozygous deletion f BIRC2(cIAP) and BIRC3(cIAP2) in primary NPC
• Identification of a novel 12p13.3 amplicon in NPC
• Cancer 2017 -May Hayek M, Issaeva N et al
• TRAF3/CYLD mutations identify a distinct subset of HPVC associated HNSCC
• TRAF3/CYLD/TRAF3
• Development of sub lines with logistics of MHC Class I expression
• Resistant to immunmotherapy?
• Somatic genetic changes in a paired primary and recurrent NPC tumors
• TLR3/IRF3/IRF7 —> NFKB
• Catalogue of genetic alterations in NPC cell lines and PDXs
• C15 LMP1 overexpression —> coupled wiht no NFKB mutation
• NFKB addiction f EBV-positive NPC
• KO of BCL3, p50 and
• BCL-3
• PI3K/MAPK activator mutations associated with poor outcome in patients with recurrent or metastatic tumors
• ARID1A mutation is rare in this cohort

2016 GRC NPC

Kwok-Wai Lo (The Chinese University of Hong Kong, Hong Kong SAR China) "NPC Genetics: Progressive Genomic Changes" 羅國煒 (https://goo.gl/gdha6A)

1. Undifferentiated NP cells abundantly infiltrated with immune cells
2. Loss of chromosome 3p is a common event in NPC
3. Ch 9p21 (p16/CDKN2A) deletion is an early event in NPC development in > 95% of EBV +ve NPC
4. All EBV +ve gastric cancers are with p16 hypermethylation
5. Singapore Nat Gene paper: WES 56 NPC normal/tumor pairs + capture-seq of 66 NPC samples. Results: low mutation rate and wide mutational diversity
6. Lo’s group: WES (111/111) and WGS (15/111) of micro-dissected EBV +ve NPC tissues (> 95% non-keratinizing cells; 78 primary, 11 local, 22 distant meta mostly from lung and liver; mean age 45-yr old)
7. By using NMF algorithm, identifying: (1) deamination of 5-methylcytosine process (2) defective DNA mismatch repair (3) high mutation load is associated with poor outcome

GRC NPC - Liu, Fei-Fei

Fei-Fei Liu (Princess Margaret Hospital, Canada)
"Prognostic Significance of miRNA Signatures for Detection of Metastatic NPC"

• metastatic NPC, metastamirs   let-7  miR9
• Nanostring 654 miRNA + 34 EBV mi RNA +   mi RNA from other viruses on 124 patients
• Bruce Oncotarget , 2015 miRNAS 154 + miRNA 449 - MENA 140 - MIRNA 34c (Bruce et al., Oncotarget, 2015)
• N stage coefficient 0.3 p - value very significant
• Adding LN status to miRNA signature
• However, no miRNA signatures overlaps with those in Liu et al (Liu et al., Lancet Oncol, 2012 馬駿group)  
• -----------以下未發表
• Cell line data NP69 NP460 C666-1     miR-499  pops out
• KO of more 499  73 downregulated ones top 1 is TGFBI
• CO-IP OF ITGB3 and TGFBI (also expressed into media)
• miRNA in TME!!
• Also exsomal miRNA in metastatic niche
• CD9, FLOT1

From Ming-Han

• @Some miRNA levels highly associated to tumorigenesis (white et al, Nature review clinical oncology 8:75 2014)
• @FFPE->microdissection-> RNA kit-> sequence.
• @654 hmiRNA; 34EBV miRNAs had been detect
• @normal NPE cell as control.
• @use combined calculation to calculate:
• miR154*0.147+mir449b*0.28-mir34C*0.311-miR140*0.653 (if value is high: high risk of relapse!!)
• (Bruce et al: oncotarget 6: 4537 2015yr).
• Especially mir449b and miR34C highly expressed in EBV+ NPC cell line.
• @Mechanisms: KD of miR34C or mir449b can make C666 sensitive to Cisplatin. (C666 doesn't affair Cisplatin!)
• What's the target?? TGFBI
• 
  
• Tumor repression
• Low TFGBI consistent to promoter methylation
• TGFBI KO mice: no tumor.
• tf TGFBI into C666: become sensitive to cisplatin.
• KD TGFBI into NP69: resistant to cisplatin.
• Use 293 IP-> flag TGFBI-> pull down ITGB3.
• TGFBI is secreting protein.
• Model: TGFBI bind to ITGB3-> Block PTEN
• TGFB-> induce PI3K---> PI3Kp-> resistant to cisplatin

	C666	NP460
pAKTser473	+++++	-
TGFBI	-	++++

They found exosomes have huge amount of miRNAs.

	Exosome	Supernatant	cell
CD9	+++++	-	+	exosome
FLOT1	+++	+	+++
GRP94	-	-	++++	ER marker

GRC NPC - Lu, Xin

Xin Lu (Ludwig Institute for Cancer Research, United Kingdom) 
"p53, a Key Regulator of Genomic Stability in Cancer"

• Discovery of p53: 1979 about the same time “tumor antigen” by Lloyd and Old, PNAS.
• Binding partner of large T antigen of SV40 (A Larid and David Lane)
• p53 mutation in 30–40% BL, why only 9 % in NPC 
• A review paper in 2008 (Murray-Zmijewski et al., Nat Rev Mol Cell Biol, 2008)
• P53: a guardian of cellular plasticity (ability to change for one state to another)
• KLF4/SOX2/OCT4/MYC → iPS. In this setting, get rid of p53 (or p16) enhances iPS.
• Over 80% melanoma cell harbor structurally wild type but functionally defective p53, why?
• Synthetic suppression (B-RAF 50%, p53 <20 150="" been="" cellular="" have="" identified="" interact="" li="" over="" p53="" protein="" to="" with="">
• ASPP (ankyrin-repeat, (Zta also has this motif?), SH3-domain)
• ASPP1, ASPP2, iASPP
• ASPP1 and 2 (陰, which activate p53/p63/p73)
• iASPP (陽, which inactivate p53/p63/p73)
• JNJ-7706621 CCNB/CDK1 inhibitor can be used as an iASPP inhibitor since iASPP inactivates p53 via phosphrylating CCNB1/CDK1. (+Nutlin to suppress MDM2)
• RaDAR (RanGDP/Ankyrin repeat binding) code (Lu et al., Cell, 2014)
• Hyrdophobic 13th residue (Notch, Ankyrin 1, 2, NF-KB etc)
• Altering RaDAR code can change cellular location of ARPs.
• CDKN2A (p16) M53I & R87P both are 13th residue of ankyrin repeats
• ARPs are enriched in the nucleus and many RaDAR code containing ARPs are regulators of NFKB & p53
• Many cytoplasmic-nuclear shuttling proteins cannot bind DNA but are able to influence transcript (MRTF → binds actin in the cytoplasm, b-catenin, ASPP, YAP etc) (Lu et al., Nat Rev Mol Cell Biol, 2016)
• 好像聽到她提到 Zta 有ankyrin motif, CYLD/BCL3/NFKB blablabla

------------------------------------------------------------------------
From Ming-Han

• P53 problem in 70% of BL; most of NPC cell line but only 9% of the PRIMARY NPC TUMOR. It seems p53 functions totally different in different cell line.
• ie: P53+53bp1: DNA repair
•     P53+53BP2; p53+BCLXL, P53+ASSP+DNA…. many different functions.
• @By ips (KLH4, Sox2, myc, Oct4); we found P53 or p16 can block the differentiation of ups cells.
• @RaDarCode (interestingly the protein that regulate p53 are more often by using this way than NLS)
• Why some proteins has no NLS but can anyway move to the nucleus?
• -example: RanGDP/Ankynin repeats binding code.
• NLS binding to importin-> RanGTP to GDP-> bring NLSprotein to nucleus  -> NES code move outside.
• Example: EBV CYLD: (CYLD+BCL3) to nucleus that can elevate p53 level But if CYLD mutant then can't move to nucleus; even if p53 is intact but similar to p53 mutation.
• 聽到有點恍神這個

GRC NPC - Futreal, Andrew

Andrew Futreal (University of Texas MD Anderson Cancer Center, USA) 
"Cancer Genomics and Evolution"

• tumors heterogeneous from one patient to another
• intratumor heterogeneity—a state where subclonal subpopulations within the same tumor accumulate different kinds of mutations over time (65% heterogeneity in RCC)
• 1 mutation/wk, 40 mutations / year
• APOLLO platform  https://goo.gl/uRmjHX
• Moon shot platform
• 40% subclonal mutation vs 17% no subclonal mutation
• Immune signature mapping (n=54) → anti-CTLA4 --responder (n=8)  and progressor (all sent to PCR-seq check for clonality), for progressors → anti-PD1 blockade → responder (n=17) progressor (n=29)
• TCR beta chain-seq for neoantigen
• Only individual biomarkers are predictive
• Mutation load and neoantigen and common drivers: gene mutations do not predict response to immune checkpoint blockade, but TCR clonality is seen in responders to anti-PD1
• Genome-wide copy loss correlates with responder (KEGG/immune response pathway loss) 這個結論與 Science 2015 這一篇一樣  (LA Garraway group (Van Allen et al., Science, 2015)
• Subclonal evolution under immune pressure (CLL & transplant), multiple rounds of chemotherapy driver genes
• Driver genes : ERG2 appears at later time / p53 also emerges after many years
• Clone T cells → do single cell seq → any special T cells responsible for eliminate some portions of tumor cells

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

From Ming-Han

Goal: Germline genomic vs extrinsic factors (microbe, chemical, environment) vs tumor heterogeneity

General: they separate cancer patients with many groups; whether they are response to anti-CTLA4 or anti-PDL1

@melanoma:

Before treatment (biopsy)-> treat with CTLA4 -> replies (biopsy) -> antiPDL1 -> relapse (biopsy) ---> sequence tumor genomic and blood components

Conclusion:

• Patients response to PDL1: high CD8+T cells, tumor express PDL1 at high level.
• If patients' clonality of T cells is higher: response to PD1 better.
• If genomic copy number tends to "loss": both CTLA4 and PDL1 work well. If genomic tends to "gain" copy: no response. 
• Loss of copy number in cancer genome tends to have the reduced immune response.

@CLL treatment: transfusion allogenic donor's T cells? (FCR treatment, DLI? WES windows?)

-allosct? DLI?

@TRA framework: the Apollo TRA team:

Try to combine the clinical and genomic and whatever together for the more specific treatment in the future.

GRC NPC - Hildesheim, Allan

Allan Hildesheim (National Cancer Institute, NIH, USA) 
"Use of Viral and Genetic Markers for Risk Stratification for Development of Screening Programs for the Early Detection of NPC"

1. 1987 Use of viral and genetic markers for NPC risk stratification.
2. 16% cancers are caused by infectious agents (Lancet Oncol 2012, de Martel)
3. EBV vaccination: EBV gp350 (Coghill et al., Clin Cancer Res, 2016 High Levels of Antibody that Neutralize B-cell Infection of Epstein-Barr Virus and that Bind EBV gp350 Are Associated with a Lower Risk of Nasopharyngeal Carcinoma)
4. Screening for NPC: (Chien et al., N Engl J Med, 2001 Serologic markers of Epstein-Barr virus infection and nasopharyngeal carcinoma in Taiwanese men)
5. Individuals positive for anti-VCA IgA or anti-EBV DNase antibodies have a much higher risk for NPC (Yu et al., Clin Cancer Res, 2011 Prognostic utility of anti-EBV antibody testing for defining NPC risk among individuals from high-risk NPC families.)
6. For those unaffected families (members from NPC multiplex families) anti-EBV IgA-VCA and IgA-EBNA1 are the best (Hildesheim, Am J Epidemiol, 2013 Invited commentary: Epstein-Barr virus-based screening for the early detection of nasopharyngeal carcinoma: a new frontier)
7. Protein array (EBV1 chip Muta, B95-8, Akata strains, Middledorf), Cohort design: NPC array project strategy. Screen for promising candidate markers using case-control design → evaluate top hits in population-based screening
8. Special notes for case-control design: estimate sensitivity and specificity, but not positivity and negativity
9. LF2 (IgG), BGLF2(IgG), Rta(IgG), BMRF1-EAD(IgA), BXLF1-TK(IgA) | two or three of them achieve highest SE and SP
10. EBV DNA viral load in plasma (insensitive for early stage NPC)
11. EBV DNA in nasopharynx swab (high specificity and sensitivity, combined with miRNA and DNA variants)
12. Host genetic risk factors: only ~7–9 % NPC is familial, which has 5-10 fold increased risk
13. Also comments on EBV lytic cycle replication in differentiated

有人踢館: 一方面anti-VCA是NPC高危險群，一方面說病人體內有anti-VCA似可neuturalize病毒、降低罹患NPC風險 這...    Allan: OOXXOOXXOOXX

From Ming-Han

• Martel; lacent oncol 2012: each pathogen and cancer: good reference!!
• EBV Vaccine (gp350 vaccine) against NPC?
• Yes this antibody can block clinical diseases of IM but not infection?
• Vaccine and childhood?
• Some data said might be work for treatment of NPC?
• The facts:
• People with NPC have higher level ab against VCA and DNase; and vice versa.
• People with NPC with much lower ab against gp350 "at the time they have NPC"; and without NPC people with high level ab against gp350. (level of anti-gp350 study not like VCA and DNase have long-term predictable results).
• If people/patient have high level anti-VCA anti DNase ab; will also have high level anti-EBNA1 abs. (Yu, Taiwan group).
• @EBV protein microarray (sensitivity; specificity problem…? It seems he has a special but correct way to determine that).
• -Use patient's serum to the microarray that had been coated with different EBV proteins to detect the antibody level.

Results:
High level of IgA against EBV-> associated to NPC
High level of IgG against EBV-> associated to lymphoma.

Top markers of antibodies in NPC high-risk or NPC patients: (all IgA)
LF2,
BGLF2,
BRLF1
BMRF1
BXLF1 (TK).

Now applying this array to BL, NKT, EBVaGC…etc.

In Aug EBV Meeting Session 10: Nasopharyngeal carcinoma and other epithelial cell tumors
Whole Exome Sequencing of NPC Multiplex Families Identifies Variants potentially Involved in EBV Control
Allan Hildesheim, National Cancer Institute, Rockville, USA

GRC NPC - zur Hausen

Harald zur Hausen (German Cancer Research Center, Germany) 
"Discovery and Early Studies of EBV in Burkitt's Lymphoma and NPC"

1. EBV provides a step in the malignization of pre-malignant NPC lesion (methylation of p16)
2. Endemic multiple sclerosis
3. TGFb is an inducing agent for EBV lytic replication
4. Vit D deficiency -> TGFb increase -> EBV reactivation
5. EBV enhances TT virus replication
6. In dually infected BL or LCL cells, EBV and BMV  bovine milk factor  --> low sun exposure/Vit D deficiency --> TGFb increases  -->  EBV reactivation --> inflammation → tissue destruction --> multiple sclerosis

From Ming-Han

• In Henle's lab before and identify EBV is specific to IM and BL (JV 1967; 1: 1174-85) and Identify abnormal ch 12. Whether EBV cause chromosome instability?
• Prove EBV associated to BL and NPC (ISH)
• NPC: epidemiological factors of NPC?
• p16 inactivation (Tsang et al) and Cyclin D1 activation (Tsang et al, PNAS 2012).
• Hypothesis: whether EBV cause NPC for the prp-malignant NP cells???
• BL: cMyc translocation is important (RAG1/2; NEHJ)
• Current research: Streker and de villiers: TT-like particles (23: 11-2006)
• Whether EBV stimulate TT virus replication? For example: CMV, Herpesvirus, adenovirus infected cells can cause lytic replication in the non-permissing cells. 
• 概念：人類的病毒在人類不會lytic但是人類病毒在動物細胞可以。反之亦然。是否動物的病毒感染人類造成chronic infection而導致MS?
• ＠發現：HSV infected HL secrete small viral particles? (Lacent 2001, 257: 381-384).
• @May animal viruses DO NOT replicate in Human but do lytic cycle in animals can cause human diseases? On the other hand, is that possible that human virus cause tumor in animals?

Model: He used milk cow viruses (episomal type; DNA virus) to test his hypothesis

• These viruses can infect human cells (RNA, protein, replication).
• Correlation: children drink milk is associating to the deficiency of VitD later, Reactivation of latent HSV infection (EBV, HHV6, VZV also), and also the high MS factor. On the other hand, if children drink mother milk>6 month and live in tropical regions may have fish factor. Importantly, the reduced of VitD is associating to the elevated MS factor.
• Why VitD and MS?
• elevated VitD3 receptor is associating to lower TGFB secretion
• TGFB elevate EBV lytic cycle.
• Whether low vitD-> big TGFB-> high EBV activation?

Result:

• EBV infection can elevate cellular small DNA virus activation!! whether these small viral particle causes chronic inflammation?
• Model: many cells infected by milk virus and many cell infected by EBV: few cells with both. these cells stimulate viral reactivation.

Q: what EBV protein cause these milk viruses from cows super replication? UNKNOWN!!!

Q: Whether these small viruses alter human immune responses? UNKNOWN and ongoing.

Q: by Rickonson: connection between infection and cancer?

A: HPV and smoker and lung cancer: actually people found 20-50% lung cancer actually not smoker.

A: Vaccine against virus: like VSV and rebulla and HPV: nearly 100% protective.

Q: EBV vaccine and NPC?

A: problem: since EBV mostly do replication in oral epithelial cells, that means the vaccine need to produce high level of AB in Saliva...

GRC NPC - Unclassified

Wed Poster #5 Dr. Timothy Wai Ho Shuen 孫傳豪	Singapore TESSA therapeutics (http://goo.gl/zdbYpA) NPC stage IVb patients → 抽300-400 ml blood → 一部分加B95-8 養成LCL → 養成的LCL與病人自己PBL aliquot co-culture → mapping 出effective CTL → expand → 打回去病人身上 (response rate還蠻不錯的! 整個時程約1-2個月)
Wed Poster #9 University of Malaya Velapasamy, Sharmila ... LF Yap	SB-431542 10 uM, a TGFBRI inhibitor. Adding of this inhibitor in NP550 or NP361 infected Akata-GFP EBV, maintain cells with higher proliferative potential!
Wed Poster #11 Whitehurst, Christopher B	BPLF1 DUB in Tegument → PCNA → interferes with Pol eta → impaired in DNA repair

---------------------------------------------------------------------------------------------------
Hong Zheng (The University of Hong Kong, Hong Kong SAR China) 
"Identification of Genetic Susceptibility Genes in Nasopharyngeal Carcinoma by Next-Generation Sequencing"

1. 67 FH (family history mean age 40), 39 EAO (early-age onset mean age 17), 59 sporadic NPC (mean age 55), 895 controls
2. Analysis pipeline (KEGG Seq and ANNOVAR)
3. Only 19 genes were overlapped between FH and EAO group
4. In the EAO group highly mutated genes (n= 157) are distributed in the following pathway: ECM, ABC, Ligand-receptor, proteoglycans, DNA replication (eg. COL17A1, MST1R, PTPRG, ITAG9)
5. Single variant association test (MST1R) Dai et al PNAS 2016 (Dai et al., Proc Natl Acad Sci U S A, 2016)

From Ming-Han

1. Phd Candidate
2. Compare three groups:
3. Family NPC (>= 2 generations have NPC cancers) Ave yr: 40 yrs.
4. Sporadic NPC (random) Ave yr: 55 yrs
5. Early-onset NPC (around 20 yr?)
6. Compare the genomic sequencing.
7. Highly mutated ones: ERCC2, FaNCA, FANCI, POLE, LIG33): all involved in DNA repair.
8. HIGHLY OVERLAY GENE WITH MUTATION: ITGA9
9. BY RARE VARIANCE?   RASSF1, TLR9, ATXN7, MST1R (Dai et al., Proc Natl Acad Sci U S A, 2016)
10. MSTR1 damage can cause tissue-specific macrophage mobility impair.

Question: If MST1R mutated: whether they have also problem with Cilia symptoms?

Question: Whether MST1R mutation cause microbe flora changes?

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

Honglin Chen (University of Hong Kong, Hong Kong SAR China) 

"EBV BART miRNA and Serological Biomarkers for NPC"

1. EBV BART  Ist paper by Griffn BE EMBO journal 
2. 2004 Science 304, p734
3. 40 mature miRNA 
4. Extracellular miR BARTs in NPC cells   extracellular  BART 3, 7,   13 JV  2014 plasma is better than serum but only miBART7 and 13 associated with NPC (not BART3)
5. Patient with metastatic NPC have high-level of BART7
6. Higher BART 7 > 5000 can distinguish outcomes

From Ming-Han

• Current biomarker of NPC: EBER, BARTmiRNA, EBVab, plasmid EBV DNA level.
• EBV miRNAs and EBV promoters can be regulated?
• @CP/LP: by EBNA2; LCL/PTLD
• @QP of EBNA1 by STAT3/NFKB
• @p1 p2 of BARTs by CEBP/NFKB
• Method: extracellular BART miRNAS (only some BART miRNAs can be highly detective)
• BART7 and 13: highly in NPC
• Plasma qPCR is more sensitivity to Serum qPCR; for miRNA.
• EBVDNA or EBV-mirBART7 which is better to predict? BART7 is better.
• Further: whether can apply serological BART-7 miRNA as an early predictive thing to guess NPC incidence? need long term research.

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Brigette Ma (Chinese University of Hong Kong, Hong Kong SAR China) 

"Novel Targets for NPC Treatment"

1. 與KW Lo, Broad Inst, UCSF, Pittsburgh U, Soul University 都有合作
2. Ref: CCR 22 1865，2016
3. Ref: Bruce  JCO Review EBV infection 
4. Potential NOTCH1
5. PDR001 NORVARTIS

From Ming-Han

• To study the effects of chemotherapy after tumor recurrence.
• Select targets->exome sequence
• NPC: few copy number change; 
• NPC: PD1 and PDL1 level (Hegde Clin Cancer research 22(8) 1865-74 2016 AACR) discuss microenvironment.
• PDL1 is up regulated in EBV+NPC (>80%).
• EBV infection elevate PDL1 in vitro; LMP1 activate PDL1.

Three drugs to test EBV:

• Pembrolizumab (phase I): specific testing for PDL1 positive tumors
• Nivolumab (phase II); this ab not cause ADCC
• PDR001 (Phase III)

Results:

• Pembrolizumab: all PDL1+ positive solid tumor; 37% not keratinized; 22% Keratining NPC
• in 67% tumor became smaller; still testing.
• Nivolumab: test only in 2 cases: tumor disappear.

Question: how to check "recurrence" after primary tumours?

1. Endoscopy; 2. MRI, 3: blood EBV DNA level But in recurrence: 60% can detect high EBV DNA whereas 40% can't. So local SWAP might be better.


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Wei Dai (The University of Hong Kong, Hong Kong SAR China) "Methylation Biomarkers in NPC"

• Aberrant methylation markers in NPC
• 450 K illumination hardshi (Chin Clin Oncol. 2016 Apr (Dai et al., Chin Clin Oncol, 2016)
• MS-HRM Int J Cancer 2014, Yang et al, a qualitative assays, needs to use positive purpose for quantification
• Illumina make this assumption when designing their array probes, ie that all CpGs in the 50bp probe sequence are co-methylated. (Eckhardt et al., Nat Genet, 2006)
• Regional analysis showed that 6p21.3 is more frequently hypermethylated.
• HLA- L TRIM31 IER3 FLIT

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提前返台、未聽到下面六個talk William Wei (Hong Kong Sanatorium & Hospital, Hong Kong SAR China) "Challenges for Surgical Management of Recurrent NPC" Raymond Ng (University of Toronto, Canada) "Detection and Screening for NPC with Trans-Oral Brushing and Determination of EBV DNA" Troy Messick (The Wistar Institute, USA) "Development of Small Molecule Inhibitors of EBNA1 for the Treatment of Nasopharyngeal Carcinoma" (Tuesday Poster # 18) Mhairi Morris (Faculty of Health and Life Sciences, De Montfort University, United Kingdom) "EBV-Encoded LMP1 Induces a Myofibroblast-Like Phenotype in Epithelial Cells via Epithelial-to-Mesenchymal Transition" (Monday Poster # 10, 二個小女生一人講一半) Alan Rickinson (University of Birmingham, United Kingdom) "Future Focus for Basic NPC and EBV Studies" Quynh Le (Stanford University, USA) "Future Personalised Clinical Care for NPC Patients"

LabLog-SFL

LogID	Title	Initial	Date
AF001	Codebook for GA341 (= YJC AE538 = YMU RNA-seq on OKB2/S1F/THP1)	SFL	10/21/15
AF002	Experimental sheme for AE592 (YMU RNA-seq GA416 and GA417)	SFL	10/23/15
AF003	Codebook for GA417	SFL	11/3/15
AF004	Codebook for GA416	SFL	11/4/15
AF005	Codebook for Po-Ting	SFL	11/5/15
AF006	Codebook for tidy 40T/N raw data (2015_1110_11SVA_40TN_raw = 40TNRaw)	SFL	11/10/15
AF007		SFL