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

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From Ming-Han

• with 6 weeks can have plasma in baby into mather.
• good example: by using non-invasive prenatal testing (NIPT) can easily detect chr21 number of baby. (yu et al, PNAS 2014; Jiang et at, PNAS 2015).
• Can use plasma DNA to predict tumours?
• Plasma DNA: short DNA fragments; nearby 150(?) nucleotides; perhaps this length is due to still binding to histone.
• Mother's DNA is longer than fetal DNA for whatever reason.
• Question: whether we can identify the DNA from different tissues? 
• Method: by using epigenetic (methylation profile). Since different tissues with different methylation profiles.
• @compare original sequence and bi-sulfate sequence.

They found:

1. Most of the plasma DNA came from liver, bone marrow and placenta.
2. by using the mixed DNA method; they can now evaluate the ratio of DNA that came from different tissues.
3. Israel groups used similar strategy but only specifically identify tissue specific epigenome for 1-2 target regions; might be dangerous.

They found:

1. Normal and healthy people; plasma DNA (50% from liver; 20-25% from lymphocytes; 20% T cells)
2. For example; if they found one individual has 60% dNA from liver or 20% DNA from B cells: highly possible having B cell lymphoma or liver tumor.
3. @ Cell 2016: 164: 57-68: prove that plasma circulating DNA actually all in nucleosome form.

To connect this technique to EBV?

1. High level in patient
2. After remove primary tumor-> within 1,5hr plasma EBV DNA drop to background.
3. After chemotherapy: after 20 hrs drop to background.
4. High level of EBV is associating to the survival.

Question: Can this techniques be used for screening EBV+tumor? 

YES! In ? Number testing sample; they found the ones with high level of EBV DNA in plasma has more than 50% cases have stage 1 NPC; XX% stage 2 or 3. Highly sensitive.

Q: method:

They choose to use W repeats as primer with the highest sensitivity but since different EBV strains might have different W repeats so need to carefully consider what's the "threshold" for normal EBV DNA screening.


2018 GRC NPC

Dennis Lo (Chinese University of Hong Kong, Hong Kong SAR China)

"Plasma EBV DNA for Screening Nasopharyngeal Carcinoma"

plasma-DNA based molecular diagnostic (liquid biopsy / CTC) fo NPC

(Original paper Cancer Research1999, Lo et al)

[characterization of plasma EBV DNA)

• size profile of plasma EBV DNA (CHan et al, Cancer Res 2003. Short nucleosome?)

• Short Half-Life. — very fast Lo et al Cabc era 2000, 4 days for CT, 1.5 h for surgery

[Early detection marker?]. Li et al. PLoS one 2014

- 70% of NPC patients were at stages 3-4 at diagnosis

- 20, 000 subjects

- 40-year-old

- Procedure. Plasma EBV DNA. —> 4 weeks —> EBV DNA test again. —> nasal endoscopy and mRI assessment

   5.5%(1,112) positive with first plasma EBV FNA

   1.5% (309) with two consecutive positive plasma EBV FNA test

   34 cases of NCOC identified (positive predictive rate: 115). [2017, NEJM publised]

~ 50 copies of EBV episodes

Each containing ~ 10 copies of PCR target

Why false positivity

— Chat et al NEJM 2018:

— Lower temperature, higher false positive

Logistics (4 weeks)

Baseline EBV DNA, follow-up EBV DNA

Study Design screening cohor 20, 174

Transiently positive

Persistent positive

True NPC

Targeted capture sequencing

Autosomal DNA

Discovery stage

— quantitative difference? Yes

– size difference. (Pregnancy test paper. Circulating 146-bp. And baby 136-bp. Without nucleosome).

Only NPC patient DNA show shorter size

EBV DBA size ratio is another important difference!!

Extrapolated. —>. PNAS paper

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