Dora L W Kwong

Lancet Oncol. 2018 Sep 10. pii: S1470-2045(18)30579-5. doi: 10.1016/S1470-2045(18)30579-5. [Epub ahead of print]

Camrelizumab for nasopharyngeal carcinoma: a new hope?

Kwong DLW1.
Author information
1
Department of Clinical Oncology, University of Hong Kong, Pokfulam, Hong Kong, Special Administrative Region, China. Electronic address: dlwkwong@hku.hk.

Wenfeng Fang and colleagues 1 report on two phase 1 studies of the programmed cell death-1 (PD-1) inhibitor, camrelizumab (SHR-1210), as a treatment for patients with recurrent or metastatic nasopharyngeal carcinoma. The first trial tested camrelizumab monotherapy in 93 patients who received at least one previous line of treatment for recurrent or metastatic nasopharyngeal carcinoma. The second trial tested camrelizumab in combination with gemcitabine and cisplatin as a first-line treatment in 23 treatment-naive patients with the same diagnosis. Both trials showed good safety profiles with no treatment-related deaths observed in patients treated with camrelizumab monotherapy or camrelizumab combined with chemotherapy. The more interesting findings are the preliminary antitumor activity of camrelizumab in both settings: 31 (34%; 95% CI 24–44) of 91 patients who received previous treatment achieved an overall response with camrelizumab monotherapy (two patients had a complete response) and 20 (91%; 77–97) of 22 patients with the combination of camrelizumab and chemotherapy as first-line treatment (one patient had a complete response). These results are the most promising so far reported for PD-1 inhibitors tested in nasopharyngeal carcinoma.

Lawrence Young

Lawrence Young (University of Warwick, United Kingdom)
"The Future of NPC Research: Opportunities and Challenges"

• Key questions 
• relevance of epithelial infection to EBV’s natural history 
• Role of EBV strain variation
• Precise contribution of EBV latent and lytic genes 
• Contribution of the microenvironment 
• Targeting EBV for ... 

Tak Mak

Tak Mak (Princess Margaret Cancer Centre, University Health Network, Canada)
"Beyond Targeting Oncogenes: Future Anti-Cancer Strategies"

Tak Mak (Princess Margaret Cancer Centre, University Health Network, Canada)
"Beyond Targeting Oncogenes: Future Anti-Cancer Strategies"

- 1983, Discover T cell receptor, 1995- published a landmark paper on the function of CTLA4

By the turn of the last century chemotherapeutic agents have hit the wall
1942 nitrogen mustard
1948
1966- the origin of cancer

Brigette Ma

Brigette Ma (The Chinese University of Hong Kong, Hong Kong SAR China)
"Immune Checkpoint Inhibitor in the Treatment of Nasopharyngeal Carcinoma: A Clinical Overview"


- The blue dots in HE (TME lymphocytes !!)
- Co-inhibitory ICPs are inductively and have immunosuppressive fun it (early: CTLA4 dampens T-cell priming; late: PDL1 act on T-effectors in TME)What’s the evidence on NPC? (2018, Duffild at al 2018, AACTR abstract 4750 —> PDL1 is expressed in immune cells + PD-L1 is induced by LMP1Fang 2014 Oncotarget 5(23)p12189
- Oncoimmunology Zhu 2017 (intratumor PD1 is important)
- Gal-9 and TIM-3 contributes to immune evasion in NPC and canc bear reversed in vireo
- NPC Exocets (C17) induces apoptosis
- Chen et al Sci Report 7, 2017 Gal-9/TIM3 and Fox3p expression (TC/TILS) in 95 paired primary and recurrent non-keratinging NPC tumors
- 2017 ASACR abstract 646 (Siu S and KW Lo —> PD1 , PD-:2 and IDO

Han Chong Toh

Han Chong Toh (National Cancer Centre Singapore, Singapore)
"Cellular Immunotherapy for Nasopharyngeal Cancer: Learning from the Past for a Better Tomorrow"


Tetrament of metastatic NPC with autologous dendritic cells transfigured with adenoviral veryor (AD5F35) expressing LMP1 and LMP2 genes in patients (NPC vaccine patient 004 -->  partial response; knowledge is of no value unless ups put it into practice Anton Chekhov).

Treatment of NOC with EPV using autologous T cell therapy
- phase II trial of GC+EBVST in advanced NPC
- Gemcitabine induces MDSCs
- T cells as living therapy (injet 10 T cells into the body can generate 3 logs T cells
- Demonstrated success in phase II solid tumor trials (Tessa’s phase II NPC trial showed improved IS rate and quality of lifet; response rate 43%
- Significant ling-term survival beyond 3 years (3-year, 37%, 4-yr 26%, 5-yr 11%)
- Strong response rate during uhmmunotherapy phass (complete response 6%, partial response 13%, response
- Spider plot of tumor sizes (Gem.carbiplatin and CTL therapy (N=20)

Graham Taylor

Graham Taylor (University of Birmingham, United Kingdom)
"The NPC Immunotherapy Landscape"


Broad CD8+ and CD4+ T cell responses to vaccination in ex Vito ELIspot immune assays
- weal pre-existing EBVNA1 and LMPO2 T-cell
- Vaccination simulates multiple CD8+ and CD4+ T cell responses within an individual; lpatuens
- Anti-tumor T cell responses before vaccination
- 95% T celll go to tissues, 5% T cell in the blood (>)
- Cultured T cell assay
- — otauent cells in 96U plate culture 10-14 days
- Responses after chmoradiotherapy , below limit of detection ion ex Vito assays can amplify after vaccination
- UK phase 1Blocker trial, 80 year kid oatuebntm remission ., Signle diodes 5x 10^8
Possible epitope spreading to non-target antigens (HK Phase II Trial, patten
- no target antigen BARF1
So far, just IFNG as a read out, this is only one dimension f data —> MHC-tetramers (MHC + 3 antigens) will add additional readout

Rajiv Khanna

Rajiv Khanna (QIMR Berghofer Medical Research Institute, Australia)
"Adoptive T Cell Therapy for NPC: Immune Correlates to Immune Contexture"

• Collaborates with Victor Lee, Dora Kwong, Hohn Nichols, Vivian Li, Daniel Chua, Randall ‘Tis Janice Tsang ... at HKU)
• 2017/10/20 second CD19 CAR-T
• NPC T cell immunotherapy in the past 10 years
• Singapore group

Lillian Siu

Lillian Siu (Princess Margaret Cancer Centre, Canada)
"Immune and Other Biomarkers of Resistance to Anti-PD1 Therapy in NPC"

PDL1 in NPC, Limitation of ORR as a surrogate of benefit with IO therapies Other buiomarkers of interest (HLA-I, TMA, immunoprofuiling, radio is signatures, microbiome)

HSC et al. PD-L1 >= 1% tumor cells or TILS (22C3) J Clin Oncol 2017
Ma et al. J Clin Oncol 20128

Troy Messick

Troy Messick (The Wistar Institute, USA)
"Development of an EBNA1 Inhibitor for the Treatment of NPC: Toward First-in-Human Studies"

• EBNA1 is master regulator of ENBV latency
• ENA1 is the only viral protein expressed in all EBV cancers
• EBNA1 is required for cell transformation and tumorigenesius
• EBNA1 structure is now and druggable
• No EBNA1 orthologs in human proteome predicates a high therapeutic index
• EBNA1 is a druggable target selected 100 fragments, soaked 5 compounds for each crystal
• Collected data
• Candidate selection campaign
• - 90+high quality structure
• Biochemical assay IC50=299 nM
• VK1727, VK
• Xenografts (NSG). VK-2019 10 mpk IP
• C666-1
• MutiLCL
• C15-PDX NPC (Morocco)
• C17-PDX NPC ()
• VK-2019 is effective in NPC-PDX

• 3 mpk, 30, 100 mpk
• Combination of VK2019 and RT (radiotherapy)
• Target engagement studies
• - TGFb are affected by treatment
• - EBV qPCR for C15-PDX (EBNA1 and Bam-HI W
• - EBER-ISH time course expertment
• - Toxicology studies prior to IND
• - Maximum tolerated does (MTD)
• - Dose range finding
• Summary
• - no toxicology findings that preclude further development
• - Observe excellent exposure
• - No severe toxicity observed
• Exposure summary

2016 GRC NPC

Troy Messick (The Wistar Institute, USA) "Development of Small Molecule Inhibitors of EBNA1 for the Treatment of Nasopharyngeal Carcinoma" (Tuesday Poster # 18)

Link to 2016 GRC NPC PA Lieberman page

Anne Lee

Anne Lee (The University of Hong Kong, Hong Kong SAR China & The University of Hong Kong at Shunzen) 

"Challenges and Novel Approaches in Radiotherapy for Nasopharyngeal Carcinoma"

• HKNPC1301 study on IMRT (2001-2010) to further improve tomorrow control for poor risk group
• IMRT: parotid sparing (randomized trials on IMRT (late toxicity following IRT)
• Radiotherapy - does response relationship
• Fundamental + Novel to achieve “personalized NPC treatment”
• Further development of IMRT
• Radiotherapy set-up with image
• Accurate delineation of GTV
• GTC; fusion of MRI and PET-CT
• Technological advance: MRI-simulation, MRI-Linac
• RT technology evolution (particle beam RT hydro therapy)
• Intensity modulated proton therapy
• IMPT vs IMRT for NPC: decreases in radiation does to normal structures 
• carbon Ion radiotherapy for recurrent NPC
• Boron Neutron Capture Therapy (BNCT)
• 
  
• Approach 1B: adaptive RT to ensure continual conformity re-planning
• re-planning in fractionated IMRT

• Approach 2A:
• marked variations in philosophies and practice ( == clinical target volumes (CTV) for nasopharyngeal carcinoma R&O 2018
• Atlas of illustrative case of a T1N1M). —> international consensus

• Approach 2B: to improve consistency and efficiency; development of artificial intelligence

• AI in precision oncology
• Nomogram fro reining risk assessment
• - Prognostic utility of 18
• - Pre-RT EBV-DNA
• - Prognostication of EBV DNA at midpoint of RT
• - ASCO 2017 (from Taiwan guideline)

• Genome/Genetic profile to predict RT and chemo sensitivity
• RSI a hub of ten genes for NPC
• Radionics
• Head and neck cancer meeting 2018 O’Suillvin et al (?)

Danny Rischin

Danny Rischin (Peter MacCallum Cancer Centre, Australia)
"Role of Chemotherapy in NPC: Not Novel, but Still Challenging"

• Discussion Leader (Brian OSuillivan): Danny Rischin is a leader of HNSCC! 哈，找了下PubMed還真的做許多HNSC相關研究 e.g.

J Oral Pathol Med. 2018 Jan;47(1):18-24. 
The role of human papillomavirus in p16-positive oral cancers.
Belobrov S1, Cornall AM2,3,4, Young RJ5, Koo K6, Angel C7, Wiesenfeld D1,6,8, Rischin D9, Garland SM2,3,4, McCullough M1.

Abstract
BACKGROUND:
The aim of this study was to identify the presence and frequency of human papillomavirus (HPV) nucleic acid in p16-positive oral squamous cell carcinomas (OSCCs), to assess whether the virus was transcriptionally active and to assess the utility of p16 overexpression as a surrogate marker for HPV in OSCC.

METHODS:
Forty-six OSCC patients treated between 2007 and 2011 with available formalin-fixed paraffin-embedded (FFPE) specimens were included. Twenty-three patients were positive for p16 by immunohistochemistry (IHC) and these were matched with 23 patients with p16-negative tumours. Laser capture microdissection of the FFPE OSCC tissues was undertaken to isolate invasive tumour tissue. DNA was extracted and tested for high-risk HPV types using a PCR-ELISA method based on the L1 SPF10 consensus primers, and a real-time PCR method targeting HPV-16 and HPV-18 E6 region. Genotyping of HPV-positive cases was performed using a reverse line blot hybridization assay (Inno-LiPA). RNAScope® (a chromogenic RNA in situ hybridization assay) was utilized to detect E6/E7 mRNA of known high-risk HPV types for detection of transcriptionally active virus.

RESULTS:
HPV DNA was found in 3 OSCC cases, all of which were p16 IHC-positive. Two cases were genotyped as HPV-16 and one as HPV-33. Only one of the HPV-16 cases was confirmed to harbour transcriptionally active virus via HPV RNA ISH.

CONCLUSION:
We have shown that the presence of transcriptionally active HPV rarely occurs in OSCC and that p16 is not an appropriate surrogate marker for HPV in OSCC cases. We propose that non-viral mechanisms are responsible for the majority of IHC p16 overexpression in OSCC.  (下回陳所長再提到此事，我就有充分證據!!!)


Back to the talk

• Chemotherapy for recurrent metastatic disease
• distant metastasis at presentation
• platinum doublets including gemcitabine not superior to other platinum doublets in lung or ovarian cancer
• role of local regional treatment in patients with
• Future trials in recurrent metastatic NPC
• - role of immunotherapy Indy active investigation
• - similar

• Chemotherapy has cp,[;ex and pleiotropic effects on anti tumor immune responses. (KEYNOTE-189 study design (NCT02578680)
• Overall survival by PD-L1 TPS
• TPS < 1%. TPS 1-49%
• Conclusions
• - despite chemosnesitivity o fnPPC paucity of randomized trials in metastatic disease
• - Gemicitable/platinum. (Gemicitabine history wiki —> originally for antivirus !?)

Maria Massucci

Maria Massucci (Karolinska Institutet, Sweden)
"A Role of Bacterial Co-Infection in the Pathogenesis of NPC?"

in immunosuppressive considerations. EBV positivity
1. Immunotherapy blasting clymohoma (60&)
    CNS localization (100%)
3. Post-transplant lymphoma (100%)
4. Lymphoma of the elderly (100%)
5. Leiomyosarcoma (smooth muscles)
[evidence suggesting the involvement of bacterial infections in NPC pathogenesis]
• NPC is often associated with chronic bacteria infections of the nasopharynx sand oral cavity
• Poor oral hygiene is a risk factor for NPC
• Bacterial-induced inflammation may alter the activation/differentiation of epithelial cells
• Bacterial metabolites can modulate

Pierre Busson

Pierre Busson (Gustave Roussy, France)
"Galectin-9 and Other Factors of Immune Escape in NPC Tumor Microenvironment: How to Bring Them Under Control?"

• Overview of TME
• [Co-evolution of tumor and stroma]M. Here’s at al, J Pathol 2013 (squamous-columnar junction cells
• From tumor micro environment to tumor macro-environment
• A gradient from tumor interstitial to the bloodstream
• Distant site: increase of Treg, NK and downregulation of CD4
• 
  
• Normal-tumor heterogeneity of the malignant cells
• Nortmoxiac vs hypnosis or necrotic
• EBV latent vs EBV lytic
• LMP10positive vs LMP1 negative

Daniel De Carvalho

Daniel De Carvalho (University Health Network / University of Toronto, Canada)
"Translational Aspects of DNA Methylation in Cancer"

• [DNA Methylation patterns are tissue specific and conserved across individuals]
• Deconvoluting the tumor microenvironment from DNA methylation data CYBERSORT to profile TME TILS
• DNA methylation patterns are reprogrammed in cancer patients but retain tissue-specificitcirculating cell-free DNA (cfDNA)
• [Cancer Cell 2014 Hoadly. ]
• Detection f ctDNA is a function of ctDNA abundance, sequencing depth and number of features investigated. (Shen et al submitted)
• Our solution -> capture-based approach
• Antibody IP + filler DNA
• cfMeDIP-seq in cell line DNA
• Thousands of DMRs
• Pancreatic cancer samples (n=400 in validation cohort)
• CfDNA methylation profile matches tumor profile 
• Hoadley et al Cancer Cell 2014. Reprogramming of Methylation in cancer
• Classifier trained in the TCGA data
• CfMeDIP-seq can discriminate different cancer sub-types

Wei Dai

Wei Dai (The University of Hong Kong, Hong Kong SAR China)
"Genetic Susceptibility in Nasopharyngeal Carcinoma"

• multiple genetic effects in the NPC susceptibility genes
• allele frequency can
• WES-seq if NPC identifies MST1R as a genetic susceptibility gene in nasopharyngeal carcinoma
• [Gene Level replicaiton study in NPC]
• NPC cases (n=1131, FamilyHIstory FH+=258)
• MST1R deleterious covariant s of MST1R in NPC
• Two transcript variants were identified, shorter one is associated with more aggressiveness

• Three cohorts
• Lin Nat Genetics
• HKU WES Zheng et al PNAS 2
• CUHK WES data Li Nat Commun 2017
• HKU targeted seq data (Zheng et am PNAS 2016)
• Dai wt al Maria Lung PNAS 2016
• Hypermethylation of MST1R promoter can be relevant to NPC FH+
• Reduced promoter activity by hypermethylation, can push for the shorter transcription variant —> which is associated with NPC aggressiveness
• pR306H variant has a more aggressive diseases

2016 GRC NPC

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

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

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

Scott Bratman

Scott Bratman (Princess Margaret Cancer Centre, Canada)
"Circulating Tumor DNA: Detection and Applications in Curable Cancers"

Can liquid biopsy replace it’s sue biopsy?
EGFR-mutant NSCLC: ctDNA analysis is standard for guiding EGFR

How about applied to cancers that are curable

CtDNA < 1% total cell DNA

Nat Rev Wan... Rosenfield et al. Nat Rev Cancer 2017

Rasta I and Bratman, Radiotherapy Oncol 2017: utilizing ctDNA for curable cancers: where is potential for clinical utility
1 minima residual disease (MRD): rise stratification after treatment
Molecular MRD analysis is

Maria Lung

Maria Lung (University of Hong Kong, Hong Kong SAR China)
"New Insights in NPC Detection Utilizing Circulating Tumor Cell Enumeration and NGS Analysis" 

  NPC AoE Research Tissue Bank (http://www.cnpcr.hku.hk/tb1.htm) 

• CTCs and CtDNA (Dr. Lin, JC and Dennsi Lo) provide different and complementary information
• Cancer Research 2013. (Pastel and Alex Panabieres) Real time liquid biopsy in cancer patients: fact or fiction
• CTC Number. Cell type
• 1-1000. CTCs
• 50 million. WBCs
• Like finding you among 7.4 million people
• Enrichment of CTCs by microfluidic device by size
• —> CK and EpCAM + CD45 (excluding lymphocytes)
• NGS-based SeqCp targeted capture approach (nimble gene)

Jin-Ching Lin

Jin-Ching Lin (National Yang-Ming University, Taiwan)
"Application of Plasma EBV DNA Assay in Daily Practice of NPC Management"

[Tumor Biomarkers in NPC]
- serum anti-EBV antibody
- Serum/plasma EBV DNA
- Others (many molecular markers reported bu no any one can be used clinically)

[Comparison of sensivity and specificity In J Radiat Oncol Biol Phys 2007; 67:130-7

[Ideal Tumor Markers]: non-invasive procedure, easy assay, tumor-specific,  highly correlated with tumor burden (stage)

Wei-Hua Jia

Wei-Hua Jia (Sun Yat-sen University Cancer Center, China)
"Genetic Predisposition of Nasopharyngeal Carcinoma: Evidence from GWAS and Sequencing Studies"

# Spatial and temporal patterns of MNOC mortality in China (1973-2005) showed significant decreased

Compared with those living in North China, residents living in Southern China had a much higher risk in the

Familial clustering has been observed not only in high-risk areas of

A genome-wide complex trait analysis to estimate heritability explained by common SNPs in GWAS array. (In T Cancer 2017).
GWS predicts 9 common SNPs

Lori Frappier

Lori Frappier (University of Toronto, Canada)

"New Functions for EBV Proteins in Manipulating Cellular Processes Important for Cancer"

• EBV lytic infection and cancer
• EBV encodes ~ 80 proteins, ~70 of which are only expressed in lytic infection
• EBV induced cancers are considered to be latent infections but lytic infection is also important because:
• 1 High viral load due to lytic infection precedes the development of NPC and
• Transcriptome data of 8 EBV positive gastric cancer
• 
  
• EBV manipulation of the DNA damage response
• Herpesvirus cell line are dsDNA genome that are sensed by the cell as dsDNA breaks
• Screening or EBV proteins that decrease 53BP1 Fock
• Identification 8 EBV viral proteins that inhibit DDR, including tegument BKFR4

Shannon Kenney

Shannon Kenney (McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, USA) "Differentiation-Dependent Lytic EBV Reactivation in Epithelial Cells"

• Two models of herpesvirus infection (B cell and epithelials)
• Latent —> Lytic
• Latent/B cells
• Few viral genes expressed
• Replicated by host cell DNA polymerase
• Episomal viral DNA helps viruses evade immune response
• EBV genome is methylated in the B cells
• Lytic / oropharyngeal
• Oral hairy leukoplakia: completely lytic replication
• EBV infection of normal epithelial cells is restricted to differentiated layers and is lytic. (Young et al, J Virol 1991)

Selected Posters

Lin Feng (Sun Yat-sen University Cancer Center, China)
–"Nasopharyngeal Carcinoma Susceptibility Gene TNFRSF19 Inhibits TGFβ Signaling and Promotes Tumorigenesis"

• TNFRSF19 susceptibility gene. Nat Genet 2010. Bei JX et al
• A orphan receptor , no ligand found yet
• by IHC, TFNRSF19 is highly expressed in NPC tissues, possibly an oncogene
• Survival TNF receptors noncanonical TNFR of TNFRSF19
• Identification of TNFRSF19 as
• Constitutive association of SF19 with TbR1
• TbR1/Smad2/3 is blocked by SF19
• TGF signal transduction without SF19 - late response
• SF19 is mainly involved in TGFb pathway
• Reverse correlation between SF10 and TGFb in NPC biopsy
• TGFb and EBV reactivation
• TGFb induces latent to lytic switch of EBV by activating BZLF1
• Smads-responsive element
• First hit (SF19, HLA, p16)
• Pre-cancer TGFb signaling decreases, Epithelial cell proliferation increases, permissive for EBV latent infection
• after EBV infection. NF-KB, PI3K-KAT signaling, cell survival, anti-apoptosis, genomic instability
• in Clinics. TGFBRi. Used in in idiopathic ___ fibrosis

Bo Zhao

Bo Zhao (Brigham and Women's Hospital / Harvard Medical School, USA)

–"NPC Epigenetic Landscapes and Super-Enhancers"

Chromatin modifying enzymes are frequently altered in NPC (Lin D et al Nature Genetics 2014) exome-seq results

Epigenetic changes are reversible and druggable

Super-enhancers are recently discovered enhancers with

Rebecca Skalsky

Rebecca Skalsky (Oregon Health and Science University, USA)
"Regulation of Host Signaling Pathways by EBV miRNAs"

EBV encodes 25 precursor miRNAs

~100-200 different target sites on mRNAs
> 44 EBV miRNAs =significan tregulatory potential


MiRNAs are great toools for viruses:
- non-immunogenicity
- -require limited encoding capacity (only 22 not)
-can rapidly evolve; have

Lymphocryptoivirus muRNAs are highly conserves

Group Photo

Bill Sugden

Bill Sugden (McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, USA) "How EBV Prevails During Productive Infection"

• Hybrid of P3HR1 and D98. (Endogenous P3HR1 cells)
• EBV’s lyticv phase commandeers the cell much as do a-herpesviruses
• Determining when vEBV DNA amplifies following induction off the productive cycle (treated cells with tamoxifen —> BZLF 1 expression)
• Tamoxifen was added before G2
• Virus waits until S phase
• Imaging of single cells for 45 house show that cells begin amplifying their amplicon DNA form 5-22 hours following induction
• Once viral DNA is made, it runs fast
• How much is viral (and cellular) DNA made
• 
  
• Real-time PCR snows that both endogenous EBV and its amplicon DNA increase by 1 100-1000 fold during the lytic cycle
• Endogenous EBV 3000-5000 / Cell. (In 45 hours). == 10^8
• Amplicon ~ 1000 /cell (in 45 hours)
• 
  
• What is limiting factors that regulate EBV DNA replication
• 
  
• (A)Label Lamine A/C (nuclear laminitis) so that the volume of the nuclei can be estimated.
• (B) regarding encapsidated viral DNA (measuring EBV virus parties for their DNase-resistent DNA shows surprisingly little EBV DNA is encapsdidated. Extra cellular visions contain 0.5-1% of amplified DNA (= very insufficient encapsidateion)
• (C) Meselson-Stahl or density shit experiments measure changes in the density of DNAs resulting from the incorporation of heavy nucleotide during DNA synthesis
• — 8h MEselson-Stahl experiments show that EBV DNA is synthesized rapidly (_90 min / duplication of the pool)
• — 20 h.
• — 30h the rate of duplication of the pool of EBV DNA has declined dramatically

• EBV initiated the amplification of its genomes asychronously during the lytic cycle
• EBV genomes increase ~ 1000 fold in induced cells
• This amplification is expotentional for ~ 13 hours and then declines
• This decline results from the rate of duplication per se of the pool of EBV DNNA declining dramatically
• Only ~ 1% of EBV’s amplified genomes are encapsidated, indicating that encapsidation does not contribute to the decline of genome amplification

Paul Farrell

Paul Farrell (Imperial College London, United Kingdom)

–"EBV Genome Sequence Variation: Geography, Function and Disease"

J Virol. 2017 Jul 12;91(15). Natural Variation of Epstein-Barr Virus Genes, Proteins, and Primary MicroRNA.   (PubMed Link)

• 138 new EBV genomes, 125 of these combined with 116 others = 241
• Analysis of 241 aligned EBV genomes in multiple sequence alignment
• Type1/Type2 variation and geographic
• What genes harbour the variation
• Genetic linkage in the EBV genome
• Gp350, gp42
• EBNA1
• Codon usage
• Heterogeneity
• Chronic active EBV infection
• Variation linked to NPC
• 
  
• Functional variation in EBV is related to disease
• EBV genome deletions in BL (about 10% of African BL cell lines
• Increase vBCL2 (BFRF1) expression from Wp
• EBNA-3B as a tumor suppressor gene in EBV
• E3B null is more tumorigeneic, DLBCL:
• 
  
• M81 EBV (Hong Kong NPC) has different properties from B95-8
• More epithelial cell infection
• EBVNA1 surviving binding
• Chronic active EBV infection
  Sequenced saliva EBV from 8 cases (NIH)
• No evidence of WZhet in saliva EBV genomes
• Most heterogenous regions linked to NPC is Zp V3
• SNPs in NPC Chinese vs Indonesia (27)
• G155391A RPMS1 (Feng et al 2015, Wu et al 2018)
• EBER2 (Wang e tal 2010)
• ZpV3 (Tong et al 2003)

• Type differences in gp42
• T1 and T2 differences also extends to Zp
• T2 is not a good transforming virus unless change EBNA2!

- Additional BS69 binding site in type 2 EBNA2
- 28 days
- BS69 protein binds EBNA2 and can acts as trnascription repressor (binds to promoter motif)
- BS69 dimerization
  MYND/PELSP, PQLSP, PxLxP
  MYND/PILFPD/PFLPPSDWY
- PLoS Pathogens (2016, e1005414)


EBNA2-EBNA3 genetic linkage (241 EBV genomes)

• EBNA2 T1 with EBNA3 T2 (212)
• EBNA2 T2 with EBNA3 T2 ( 22)
• EBNA2 T1 with EBNA3 T2 (  2)
• EBNA2 T2 with EBNA3 T1 (  0)
• Type 2 EBNA2 mutants work better with type 2 EBNA3
• 
  

-miRNAs

EBNA1
- DNA replication
- plasmid maintence
- transcriptional enhancer
- NOX (ROS)
- USP7 (p53, MDM2)
- Survivin (apoptosis)

- differences in codon usage between latent (more uniform) than lytic genes (codon usages more variable)
- NKTLY97.1 contains a mixture of EBV genome, the major sequence is rearranged

Irving Weissman

Irving Weissman (Stanford University School of Medicine, USA)

"Normal and Neoplastic Stem Cells"

• Marrow or MPB (mobilized peripheral blood)
• Mixture of cells: HSCs, T cells, cancer cells
• Deplete mature blood cells by labeling with magnetic antibodies
• Pure HSCs for transplantation
• 250,000 fold depletion an ever cells or T cells
• Unwanted cells : cancer cells, T cells
• Noise HSC: ckit_sca1+150+34-flk2-48-Lin-
• Human HSC: CD34+90+38loLin-45RA-
• 
  
• The problem (rescue blood system with un-purified graft giving cancer back)
• The solution (rescue with purified cancer free stem cells)
• Blood stem cell only come from blood
• Estimated MBC remaining after chemo ~ 1M; in models anti-CD47 clears these
• HoxB5+LT HSC are attached to bone marrow venous sinusoid always endothelial abluminal surfactant; 100 niches in a mouse femur
• http://symposium.cshlp.org/content/81/1.long
• Blood forming cells are attached to the bone
• Leukemia cells in AML patients
• Cell of origin - progression to leukemia. [competition!)
• Three labs’ work. Sic Translation Medicine 2012 4(149):149ra18
• (Steve Quake, Ravi Majettic, Irv Weissman)
• Identification of somatic mutations by exosome sequencing
• SKP2
• TET2 (Y1649 stop). Demethylation. CTCF FLT3 leukemia
• Ryan Corces, Ravi Mejetui and IW. 2014 PNAS (21 AMLs)
• ** if you want to know which genes are likely oncogene, ask the cancer
• Lessons for leukemia and perhaps all cancers

Patrick Moore

Patrick Moore (University of Pittsburgh, USA)

 "Why Do Viruses Cause Cancer?"

PubMed Link (pdf 2803)

Viruses do not “mean” to cause cancer

• Cancer endangers virus as well as host

• Viruses non-transmissible in tumors (eg latency, pseudo-latency)

• Virus populations

Limited resource hypothesis: enhancing lyric virus replication

tumor virus targets at G1 phase

Infection is responsible for ~1 in 5 human cancer cases

2018/06/28 Journal Club

Clinical responses to adoptive T-cell transfer can be modeled in an autologous immune-humanized mouse model.

(PubMed Link)

0621-0622 大會報告

Day 1
院長致詞: 十一月份還會有一個retreat

Emerging Trends and Opportunities for Microbiota Research
高承源副研究員 (免疫醫學中心)

- Microbiota is not just about bacteria, viruses, fungi are also included.
- Analytic tools of microbiota (e.g.assembling the human gut Microbiota in metabolic diseases.

- 16S/26S RNA sequencing)
- Metaproteomics workflow
- Models for human microbiota research
- Organ-on-a-chip RootChip (different levels of oxygen at different layers of matrix)
- Ex vivo models for microbiota research
- Experimental approaches for defining functional roles of mirobes in the human gut (PMID: 24024637)
* lost of function
* gain-of function approaches

- Determining causality in animal models
- From human fecal to mice
* germ-free mouse facility in NLAC: isolator
* Isolator at cage level

* Isocage 正壓型等級小鼠飼養機
- Natural product...
- The progression of microbiota research
Observation --> Correlative --> Causative (germ-free mice) --> Mechanistic (still very rare studies)

Microbiome projects
* US NIH HMP type1, type2 diabetes
* The earth microbiome project is. A systematic attempt to
* France
* China
* Crowdfunding microbiome project
* (UCSD) American gut
* Taiwan gut citizen science project
- Microabiota is the new key to finger many diseases
- Gordon lab.: the father of microbiome
$ Yin-yang of obesity ion the gut microbiota
$ an obesity-associated gut microbiome with increased capacity for energy harvest (Nature)
$ Causality: gut bug transmitted obsity 2013/Science
- A microbial protein that alleviates metabolic syndrome (Scientists at Belgium) A.muciniohilia (Akk bacteria)
- Dusp6 knockout mice
- CTLA4 blockade relies on gut microbiota
- How the Microbiota modulate chemotherapy and
- Gut Microbes can shape response to cancer immunotherapy
- Three papers in Science (end of 2017)
- FMT ——>

### Non-antibiotic drugs have effects on gut Microbiota
Nature article: extensive impact of non-antibiotic drugs on human gut bacteria
ClinicalTrials.gov
## Bugs as drugs ## Drug the bugs
## bacterial metabolites ## new biomarkers

- Personalized therapies & precision medicine
# guys bacteria can stop cancer drugs from working

- The microbiome understood by AI
- Microbiota research is multidisplinarity
- Key players in the microbiome (Gordon Lab, Belgium, China, Germany, Sweden)
- Synthetic and metablome
- Computational
- Metabolism and inflammation

2016/May US White House —> The microbiome Project

The most needed Taiwanese microbiome projects
— baseline studies
— healthy subjects
A potentially unique yet important baseline study project tin Taiwan

Fecal Microbiota Transplantation (FMT) for Clostridium Difficile Infection and Other Diseases  邱政洵教授 (林口長庚醫院兒童感染科)

2007年起 各國已陸續投入經費支持大型微生物菌相研究計畫

人體微生物菌相 Microbiota 人體第二基因體Microbiome

Microbiome refers to the collective genomes of these microorgamnisms

Munich of understanding of these bodily inhabitants has been derived fro 16S rRNA and metagenomiucs sequecning

Gut contains 1100 prevalent sleepiest and at least 160 species per individual

Human microbiota estimates 109-01009 trillion microbial cells, and the intestine accounts fo the largest and most diverse populations

FMT

— administration o resolution of fecal matter from a do not into the intestinal retract of a recipient to directly changes the recipient’s microbial composition and confers a health benefit

— the first FMT was developed in 1940

— rCDI (C.difficile infection) recurrent C.difficile infection

Research in CDI” Cell 2017 169, p375

• a toxin mediated infectious disease

• Immunotherapy

• Bezlotoxumab for prevention f recurrent C.diff infection (NEJM 2017)

• FMT for rCDI

— most clinical experience with FMT derived form treating recurrent or refractory Clostridioim difficile infection (rCDI)

   Quality of evuduenceL high

   Strength of recommendation L strong

Tow I open-label RCTs 94% and 90% vs vancomycin 31% and 26%m respoectiuvelky (NEJM, AP&T)

A larger double-blind RCT..

NEJM 2013 Jan 31 (compare FMT and antibiotics therapy)

  • improvement in the microbial diversity,m which persisted over time

  • Increase in bacteriophages species and Clostridium clostridium IV and XIVa (Firmicutes), and decrease in Proteobacteria

Sterile Fecal Filtrate for rCDI

• stool collected from 5 donors selected by the patients, and fully characterized according to FMT standards

• In all 5 patient, FFT restored normal stroll habits and eliminated symptoms of CDI for a minimum period of 6 months.

——-

Effect of oral capsule- vs colonoscopy-delivered FMT on rCDI (JAMA 2017)

Stool donor and FMT manufacturing pools of 7 healthy donors

• secondary outcome changes in quality of life

• minor adverse events 5-10% (nausea,

• cost capsules (308 USD) versus colono (708? USD)

IBD and IBS (still in investigation stage)

inflammatory bowel disease and inflammatory bowl syndrome

IBD may need multiple transfusions, and has donor effect

IBS placebo effect is required

FMT in the management of IBD, a systematic review and meta-analysis

FMT induces remission one patients with active UC, a RCT

• 75 adult active UC on stable does poses of IUM randomized org weekly FMTs or water enemas x 6 and evaluated for remission (...

Microbial analysis

• an increase in bactericides and proteobacteria

• insole delivery of high-diversity FMP by colonoscopy for active UC

Metabolic syndrome

• FMT in metabolic syndrome should only be performed in a research setting

Microbiome and fish for metabolic syndrome

• not all the published evidence is consistent with the hypothesis

• Much of the literatures are

Multi drug resistant disteas

3 high priority bacterial

Usding FMT to “decolonization” of MDR

Impact of DMT on complete and partial antibiotic resistant bacteria (ARB) decolonization of

Adverse events after decal microbiota transplantation (minor)

ICI (pre-clinical) immune checkpoint inhibitor

Gut microbiome influences efficacy of PD-1` based immunotherapy atainst eputhelial tumors

Enforcement policies

Therapeutic

15-30% refractory fby conventional antibiotics therapy

Vs

FMT: ~100% efficacy in the first 6-month, after 2-years still have > 80% efficacy

Taiwan FMT Expert Consensus
吳俊穎主任 (臺北榮民總醫院 轉譯研究科)   微菌叢植入治療

1st Characteristics

   First Asian FMT consensus

      Local is important (在地化)

 2nd characteristics

      Multiple stakeholders’ consensus (scholars.)

3rd characteristics

      Quality assurance by government regulations (special regulations for FMT)

Clinical Trial FMT (fecal microbiota transplantation)

台灣微菌聯盟

Steering committee 2017,/7/18

Major references

 Clin gastroenterol hapatol (US FMT workgroup 2011)

 J law and biosciences (ioenbiome 2015)

 ..

 .

 .

 .

 會議內容

2017/09/30 steering committee

基本資料及問卷

Super-donor criteria

2018/2/24 FMT consensus meeting

醫療法第62條

(5) 植入方式

A

B 經大腸鏡 將微菌叢由結腸植入

C

D

E

More AE in the colonoscopy FMT cohort

  Abdominal pain, blood in stool, FMT material leakage

Industrila developed FMT capsiule

Seres Therapeutics: SEWR 109

• cohort 1: SER-109 for 2 days (1.7 x 10^9 spores)

• Cohort 2:

Conclusions

• FMT via capsule is effective as FMT biz endoscopy delivery

• Both frozen and

• must be handled in BSL2

• Fees

  ==

• Registration

 A

 B

 C

• other diseases suitable for FMT: IBD, resistant bacteria’s, obesity, neuropsychiatric disorders, metabolic syndrome

Gut-brain Axis: a Fundamental Mechanism Modulating Alzheimer’s Progression
莊志立研究員 (分子與基因醫學研究所)

2012 Cell host and Microbes

Infection-induced intestinal oxidative stress...

Neurologiocal diseases may be affected by intestinal microbiota

Immune hemocyte (invertebrate) = plasmatocytes (verterrate)

Enteric infection facilitates immune cells mobility

FAK activity is increased in recruited or circulating immune cells upon enteric infection

Bioinformatics in Microbiome Analysis
廖玉潔副研究員 (群體健康科學研究所)


Metagenomics: the term metagenomics has been defined as

The study of DNA from

The study of DNA from uncultured organisms” (Jo Handelsman). A genome is the entire genetic information of one organism, whereas a metagenome is the entire genetic information of an ensemble of organisms. It is a culture-independent tool for studying bacterial, viral, fungal or eukaryotic communities and can be divided in two main categories.

 http://www.seqomics.hu/index.php?option=com_content&view=article&id=44&Itemid=139

Targeted amplicon: 16S

> 97% identity : same species

RDP classifier:

Silva nags

Database (GREENGENES)

TestPrime (primer design) —> a good site for primer design

An evaluation of the accuracy and speed of metagenomics analysis tools (Scientific Reports 2016 Jan 18)

Centrifuge: (Genome Medicine)

We have built an anlystical pipeline for metagenomics analysis

KEGG profiles. (Brite KEGG). Metabolites

Metagenomics assembly

 Thosands of microbial genomes shed light on interconnected biogenchemical processes in an

 • 40G /sample (current cancer metagenom ~ 5G/sample)

DrVM: detect and reconstruct viral genomes from metagenomes

Simulation datasets

10X to 50X HCV: 485-2425 100-0bp paired-end HCV reads + 3.5

Human Papilloma virus

Day 2

Opportunities and Challenges of Current Immuno-oncology Landscape
林文傑助研究員 (免疫醫學中心) 

Slide全換成和高承源一樣的、overview型態

Immunotherapy with SurVaxM fo ra patient with glioblastoma

IO global market

IGCG international cancer genomics consortium (25.

Oncolytic Virotherapy for Making Cold Tumors "HOT"
徐祖安研究員 (生技與藥物研究所)

改題目了！ —> Oncolytic Viruses: updates on current and promising landscapes

Several key factors contributing to ICI (CPI, checkpoint inhibitor) therapy failures

• low mutational loads and poor tumor immunogenicity

• immune suppressive micro environment

ICP34.5 deleted HSC with enhanced oncolytic, immune stimulating and anti-tumor properties RS Coffin Gene Therapy 2003, 10, p292

Use of new HSV strain JS1

deletion of ICP47

Deletion of ICP34.5

Earlier insertion of US11

Insertion f human GM-CSF

BioVex. company Feb 1999

Founders: Davis Latchman and Robert Coffin

CEO: Garett’s Beynon

Employees: 35

Financing to date: $20 millions

2011 BoVex was sold to Amgen

2015 JCO

Talimogene laherparepvec (T-VEC)

Anti-tumor response of T-VEC

2015 Oct T-VEC, Imlygic

local effect: tumor cell lysis

systemic effect: tumor-specific immune response

Oncologist cvirotherapy promoes intratumoral T cell infiltration and improves anti-PD1 immunotherapy (Cell 2107)

ORR=62%

ClinicalTrials.gov. 37 studies found for T-VEC

Study design and clinical responses

Combination f T-VEC and permsrolizuyman is effective i

n patients with low tumor CD8+ density

Future perspectives

• there is a limitation with regard to the degree of efficacy seems in the more advanced patients with extensive visceral disease

• The fact that T-VEC is injected into

Replimune, headquartered in Oxford UK and with a significant

RP1 anti-CTLA-4 and anti-PD1 were inserted into oncovirus

Experimental therapy of human gloom a by means of a genetically engineered virus mutant (Donald M Coen)

What else?

   RNA viruses

PVSRIPO —> poliovirus

   • Internal ribosomal entry site substitution eliminates neurovirulence in inter generic poliovirus recombinants 1996 PNAS Vol 93 pp2370-2375

   • The poliovirus IRES was replaced with an IRES from human rhinovirus. THe elimination f the neuro cytopathic phenotype was achieved.

   • Intergeneric poliovirus recombinants for the treatment of ...

Measles virus

• development of new therapy for canine mammary cancer with recombinant measles virus Molecular Theray-Oncolytic virus

Merck Viralytics. (Coxsackievirus)

Zika virus has oncolytic activity against glioblastoma J Exp Medicine. 2017 Sep (Chheda MG)

Development and Opportunities of Antibody-based Immunotherapeutics in Mainstream Oncology 官建村副所長 (生技中心生物製藥研究所) (Duke University)

CAR-T therapy is imilar to an autologous BM transplantation

CAR therapy is at the same time cell therapy, gene therapy, and immunotherapy. It represents a radical departure from all forms of medicine in existence

Tumor antigen specific gene therapy nu gene-modified T lymphocytes

• HLA-independent

• amplification of

• 41BB —> CD137-CD3 eta??

Engineered T cells for cancer therapy. History

• expression of immunoglobulin T cell receptor chimeric molecules as functional receptors with antibody-type Rosenberg

A cure for cancer

How CAR-T therapy Is reviltiozing oncology

There ar Enoch alost 300 CAR-T clinical trials running and the first CAR-T

CRS; cytokines release

Severe side effects in selected

CS19 is an ideal tumor target

• CD19 expression is restricted to B cells and possibly follicular dendritic cells

• CD19 is not expressed on pluripotent bone marrow stem cells

• CD19 is expressed

Issues in CD19 CAR-T cell therapy

Over immune reactions (cytokines release syndromes, CRS)

   Active surveillance

   Anti-IL-6R

   Steroid

   Alternative: treats patients with lower tumor burden

CND neurotoxicity

   Solution

   Tissue-specific CAR-T cell activation

Lesson learned: CD19 CAR-T cell therapy

Tumor tamed by transfer of one T cell

Dusruotiuon of TET2 promotes the therapeutic efficacy of CD19-targeted T cells(CD19 escape)

1st Gen CAR

    T cells died in 1 week

2nd Gen CAR

Largest challenges

CRS: to decrease toxicity (not meets to treat CRS bout to prevent it

Solid tumors (

Effector cells and target cells

EGFRvIII

Clinical development of RIT (MR1-1)

Directed to EGFRvIII

Dual-specific recombinant immunottoxin D2C7

D2C7-IT is an immunotixunb targeting both types of EGF receptor

EGFRvIII-CD3 bispecific antibody

EGFRvII murineCAR retroivurakl gebe delivery to marine T cells confers antigen-specific activityadpotive T-cell therapy with EGFRvIII mCAR T cells persistence

Rational development and characterization of humanized anti-EGFRVIII variant II chimeric antigens receptor T cells for

(NCT02209376)

• phase 1 : no CAR was observed

                   But T-reg was enriched

• 7x19 CAR-T

 L7/CCl19-producing CAR-T cells. Improved T cell expansion land survival during in vitro

Complete regression of pre-established solid tumor

Novel strategies to enhance the efficacy of CAR-T cell therapy for aiolid tumor

— combination therapies

Bi-specific antibodies

Dynamic Interplay Between Tumor Cells and Macrophages
楊慕華副校長 (陽明大學)


Polarization of macrophages

EMT transition Lin cancer metastasis

Science 2013 342: 708 Neto et al

Hybrid EMT. Mesenchymal cell proliferates slower than epithelial cells

2014 Cancer Cell Hsu et al 26:p534

The EMT transcription factor Snail: acetates snack poteuintiae macrophage recruited to

Snail-expressing cancer cells ar also shale to polarize macrophages toward M2. (CCL2, CCl5)

MiR-21 promotes M2 polarization of macrophages

——- Epithelial plasticity in cancer metastasis

Hybrid EMT/MET

Characteristics of

Primary TAMs express both M1;M2 markers and metastatic TAMs express M2 markers

MetastatjcTAM

Metastatic TAMs promotes mesenchumal-0epithelial transition f cancer cells

JAK-STAT6 mediated M2 mTAM induct nmetastatic colonoizatino and MET

*Ruzolitinib inhibitor. (GATA3 is an important transcription factor for epithelial cell differentiation

IK35 is an IL12 family’s cytokines produced buy regulatory nit mot effector, T-cells and Paul’s a role in immune suppression

It us a diuretic protein composed of IUL12a and IL27b chains which ar e encoded by two separate genes called IL12A and EBI2

MTAMs secrete IL23

Neutralization of IL35 represses metastatic colonization

Nature Comm 2018 Lee et al

STING and cancer

Interplay between CSC nad TME

Opportunities for Small Molecules in Immuno-oncology
顏婉菁研究員 (生技與藥物研究所)

Factors to consider for success

Antibodies vs small molecule immune checkpoint inhibitors

• effective population specific to target of interest

• PK ling t1/2 (> 15-20 days)

• co-targeting PDL1 and VISTA (v-domain Is suppressor of T-cell activation)

• PDL1 and VISTA pathways ar independent immune checkpoints that negatively regulate T-cell functions\\

• CA170- the first in class dual PPDL1/VISTA inhibitor

•

Antagonism of PDL1 and VISTA by CA170 ex-Vito activation of Hyman PMBMC

In Vito efficacy evaluation and effect on immune profiles

Oral bioacvailability in multiple species

B16/F1 tumors

Phase 1a trial: predicable dose exposure and T cell activatiton

RORr agnoists (retinoids acid receptor related orphan receptor gamma is a nuclear receptor of NR1 family

Modulate game expression in pathways that enhance immunity and decrease immune suppression

Acts as master

LYC-55716 is a selective RORg small molecule agonist

Ex Vivo T-cell assay

MC38 colon cancer: xenografts model

4T1 breast cancer xenografts model (very immunosuppressive cancer cell made)

Immuneprofile

Actvates IL17A and suppresses

Factors considered in tumor selection

* RORg expressing

* IO proof of concepts (TILs, mutation burden

* Tumor targets

Many molecular targeted agents easer immunomodulator functions

Imatinib also inhibits IDO

All inhibitors co-targeting Timor and tumor immune microenvirom,ent

Over-expression of Axl in tumors is associated with poor prognosis,, tumor progression , metastasis,m EMT transition and drug resistance

All serves as a negative regulator of inflammation and the anti-tumor I’mue response

BGB324 bemcentinib : first in class selective Axl inhibitor

MDA-MB-231 invasion inhibition

B6 mice

T-cell dependent inhibition of immune profiles

NSCLC. Axl1 high and PDL1 low

Divers immune regulatory effects of small molecules on tumor immune micro environment

   Many small molecules have direct and indirect effects on immune cell

   functions

Combination strategies wiht current checkpoint inhibitors

Translating preclinical models to clinic murine and human immune system is not comparable

Potential reasons for trial failure

• unknown MOA of IDO inhibitor I ncombination with checkpoint inhibitors

• Complex ummunoregulatory effects of small milecules

• Lack of biomakkers to monitor ummubne status for patient selection

T cel infiltration is I’portant to understand responders vs non-responders especially when targeting immune suppressive tumor microenvironment

•

Differences in trial setting small phase 2 single arm trial vs large randomized phase 3 rial

Potential reasons for trial failure

Anti cancer flavonoids are mouse selective STING agonists

(One amino acid makes a gib difference!)

New approaches for small molecules-based immunotherapy

## identifications pathways and MOA of immunosuppressive and non-immunogenicity tumor types that can only be targeted/optimized by small molecules

## Phenotypic drug discovery method

  • drug screening aims to quantity a phenotype or pathway where lknpwledge of the molecular target is nor trueired

## development of new milecular clases

  Synthetic antibody mimetics

    Small milecules with targeting and effector funtuonof the antibody

    Small meals (3-20 kDa), better solubility, tiusue penetration , stability

    SyAm-Ps:bind to prostate-specific membrane antigen and to Fc-gamma I to promote proinflammatory respinses against tumor cells

    Potential for development of nex generation customized immunotherappies

Mitochondrial DNA Leakage and Inflammatory Cytokines Activate Innate Immune Signaling and M2-macrophage in Tumor Microenvironment
李岳倫副研究員 (癌症研究所)

*The stress phenotypes in tumor microenvironment
• hypoxia • ROS • Genome instability • Acidosis
* Tumor microenvironment targeted therapies

Mitochondrion
• mitochondria can perform multiple cellular functions including bienergy production metabolism, Ca2+ signaling, apoptosis, and autophjagy
• Mitochondria are a major source or ROS in cells
• mitochondrial respiratory complex proteins are a major source of cellular ROS
• Miochondrial resporatory

* Mitochondrial Lon-induced oxidative stress in tumorigenesis
* High ROS level as a stress phenotype in tumor
* ROS are crucial signaling molecules of signal transduction pathways that promote proliferation , inflammatory,
* Lon overexpression induces. NF-KB and IFN pathways
* Lon overexpression induces the expression of IL6 IL1B IL4 and IL13 (NF-KB directed)
* Tumor associated inflammatory cytokines regulate macrophage function
* Increased Lon expression is involved in the differentiation and activation
* The effect of Lon-overexpressing cancer cells on macrophage
* OECM1
With Lon-overexpression or shRNA of Lon
* Lon overexpression —> mitochondrial DNA —> STING, IFN, NF-KB
* Innate immunity is involved STING-TBK1-IRF3 in HSC3
* Cancer Research (2016) STING promotes the growth of tumors characterized
* KO STING (in HSC3??? )decreases TBK1, IRF3/7, IDO1/PDL1


Taiwan Cancer Moonshot: Pathway to Next Generation Cancer Precision Medicine
陳玉如所長 (中央研院化學研究所) 

–Protein network critical regulation in cellular function and diseases
• Abnormal protein network is often the cause of disease initiation and progression
• Substrat, site-specific PTM regulation?
• Pathways crosstalk?
• Heterogeneity at individual patient level

–Mass spectrometry-based strategies: membrane proteome and modification

–New proteomics strategies or membrane proteome and post-translational modification
Membrane proteomics, phosphorylation, nitrosylation + others, glycosylation

–Application of proteomics strategies for delineation of disease/biology mechanism

–Nanoprobe-based affinity mass
Revised chemistry for phosphoproteomics by immobialized metal affinity chromatogranphy (IMAC)

–Ph / acid controlled IMAC 90% purification specificity and recovery

–Sequencing fractionation by IMAC-stage tip (Ti 4+, Fe 3+ Ga3+)

–Mass sepctrometry revealed dynamic interplay of phosphorylation and O-Glunacylation to switch B cell activation apoptosis. (Kudos-I Lin, Na Comm 2016)

– Lsp1 dynastic phosphorylation and O-GlcNacylation of Lsp1 switches B-cell activation and apoptosis

– Quantitative depiction of precise in biology Stoichoimetry

Phosphorylation stoichiometry is it significant

Two-fold regulation of suite stoichiometry could result from either fractional occupancy changes of 10 to 20% c=or 50 to 100% which likely represent

Are we able to differentiate changes in eithe rprotien or phosphorylation stoichiometry??

Example for phosphorylation stoichiometry measurement

The measurement of phosphorylation of

Identifying drug-resultant target through phosphoproteomic network of EGFRVIII and CK2 substrates

On the comparison between the TKI-sensitive and resistant lung cancer cell
More dramatic chang at phosphorylation level : in the resultant lung cance cell, the phosphorylation HMGA1 KD could reverse drug resistance in NSCLC PTM is important for drug resistance

Dynamic stoichiometry of EGFRVIII signaling pathway

———————-
Precious biology

An informatics-assisted mass spectrometry approach for personalized timmues membrane protein is

STOML2 as a noninvasive serological biomarker candidate fo prognosis and early CRC diagnosis

1452 origin were identified

3-protein candidates for early detection f gastric cancer. (197 protein membrane proteins)

Cancer Moonshot

US NCI 2006. Tissue Proteogenomics

CRC Nature 2014.

Proteomics identified new subtypes in colorectal cancer stage III,.. new molecular subtype


MOU signed in 2015 July
Cancer Moonshot announced by Obama 2016 Jan
VP Biden announced international al cancer Moonshot 2016, Sept)
Cancer Moonshot was included in national policy program (2016, nov) (生醫產業推動方案

2016 Taipei HUPO

International cancer proteogenome consortium

Genetic background and environmental factors contribute to unique feature of cancers in Taiwan/Asia

NSCLS

Early stage patients (n=200)
Late stage patients (n=200)

First high multi-om is atlas of personalized Taiwanese cancer cohort

Lung cancer n=400 breast cancer n=400
CRC n=400

Acre

Which platform can provide biological content to address. The unment clinical needs]]proteomics > 10,000
Phosphpoproteimics > 26000 phosphorylation sites


Most proteins in NSCLC pathways (94%) were identified

569 kinases were quantified in NSCLC tissues

Identify low abundant protein tyrosine phosphatases

Does gene subtype accurately describe a patient at the (phosphatases)

Allied specific expression profiling in lung cancer

Personalized profile in NSCLC pathway

39 SNV of somatic mutation identified in 44 patients

Night mutation rate in Asioam female (58%)
Sensitive to TKI with Del 19 and L858R mutation develop resistance within 12 moths

Personalized proteogenomic atlas revealed differential activation of signaling pathway

Gene X
Germinline mutation
Up-regulated protein level in 90% patient

X signaling and EGFRVIII-pathway (243 proteins0
Differential activation in NSCLC subtype

Degradation of extra cellular matrix

B cell receptor signaling by interleulinw

M


Taiwan protein project (2016-2019)
Applying state of the art proteogenomics to rapidly identify unique targets and pathways of cancer for detection and intervention


Targeting MCT-1/IL-6/IL-6R Signaling Inhibits EMT Progression, Cancer Stemness and M2 Macrophage Polarization in Aggressive Breast Cancer 徐欣伶副研究員 (分子與基因醫學研究所)

MCT1DENR-MCT1 heterodimer promotes ...

PUA domain a highly conserved motif in ...

Translational control of cancer (cancer translatome)

MCT1 abundance in intra-ducal and invasive breast carcinoma (IHC staining)

MCT1 overexpression associates with worse clinical outcomes indifferent type of breast cancer

MCT1 induces normal breast cell proliferation and causes malignant Alina morph Jolene’s is

CD163 as M2 macrophage marker

In vitro THP1 polarization M2 marker Arginase 1 , IL10?

qRT-PCR confirmed by using CD163, CD206 primers

MCT1 stimulates MCP1 GMCSF and IL6 secretion cytokine array. <— highly likely to involve M2 differentiation

Tocilizuman (Actemra) a humanized recombinant IL6R mAb

Discussion Session

Time	Topics	Speakers
13:30~13:40	Opening Remarks 梁賡義院長
Microbiome		Moderator	蔡世峯處長
13:40~14:25	Emerging Trends and Opportunities for Microbiota Research	高承源副研究員 (免疫醫學中心)
14:25~15:10	Fecal Microbiota Transplantation (FMT) for Clostridium Difficile Infection and Other Diseases	邱政洵教授 (林口長庚醫院兒童感染科)
15:10~15:55	Taiwan FMT Expert Consensus	吳俊穎主任 (臺北榮民總醫院 轉譯研究科)
15:55~16:20	Break
16:20~16:50	Gut-brain Axis: a Fundamental Mechanism Modulating Alzheimer’s Progression	莊志立研究員 (分子與基因醫學研究所)
16:50~17:20	Bioinformatics in Microbiome Analysis	廖玉潔副研究員 (群體健康科學研究所)
17:20~18:00	Discussion Session
18:00~	Dinner

107年6月22日 (週五)

Time	Topics	Speakers
Cancer Immunology I		Moderator	司徒惠康副院長
09:00~09:45	Opportunities and Challenges of Current Immuno-oncology Landscape	林文傑助研究員 (免疫醫學中心)
09:45~10:20	Oncolytic Virotherapy for Making Cold Tumors "HOT"	徐祖安研究員 (生技與藥物研究所)
10:20~10:30	Break

10:30~11:15	Development and Opportunities of Antibody-based Immunotherap eutics in Mainstream Oncology	官建村副所長 (生技中心生物製藥研究所)
11:15~12:00	Dynamic Interplay Between Tumor Cells and Macrophages	楊慕華副校長 (陽明大學)
12:00~13:30	Discussion Session & Lunch
Cancer Immunology II		Moderator	施修明所長
13:30~14:00	Opportunities for Small Molecules in Immuno-oncology	顏婉菁研究員 (生技與藥物研究所)
14:00~14:30	Mitochondrial DNA Leakage and Inflammatory Cytokines Activate Innate Immune Signaling and M2-macrophage in Tumor Microenvironment	李岳倫副研究員 (癌症研究所)
14:30~15:15	Taiwan Cancer Moonshot: Pathway to Next Generation Cancer Precision Medicine	陳玉如所長 (中央研院化學研究所)
15:15~15:45	Targeting MCT-1/IL-6/IL-6R Signaling Inhibits EMT Progression, Cancer Stemness and M2 Macrophage Polarization in Aggressive Breast Cancer	徐欣伶副研究員 (分子與基因醫學研究所)
15:45~	Discussion Session