Molas Analysis on Arecoline-Treated HOK Cells

02/16/2015

MTHFR, SHMT1, SHMT2

---

02/17/2015

1. Library comparison

2. OKB2/AC (>=0.1) combined with OKB2/untreated (>=1), >= 2-fold (2886 transcriptid), sent to GO

Biological Process (BP): GOBP

Molecular Function (MF): GOMF

Cellular Compartment (CC): GOCC

---

03/28/2015

Excel 製圖時

1. On "OKB2"  Filter > 3, shade them

2. On "Fold OKB2" Filter >=1.5 shade with red; Filter <=0.67 shade with blue

3. On "S1F"  Filter > 3, shade them

4. On "Fold S1F" Filter >=1.5 shade with red; Filter <=0.67 shade with blue

5. On "THP1"  Filter > 3, shade them

6. On "Fold THP1" Filter >=1.5 shade with red; Filter <=0.67 shade with blue

---

04/02/2015 

感謝大家的製圖與整理!   再來要做啥?  我有幾個想法

1. Nucleosome assembly的原料, i.e. histones, 全面下降. 表示細胞若正在S phase, 再來的nucleosome assembly將有巨大變化. 是符合hypomethylation嗎?

2. 我們map出來的histone gene多在ch6, 少數幾個在ch1. 這些cluster在一起的gene family, 使要被調降的?

3. 還有沒有其它類似例子?

4. 如果ROS是元凶 (許多AC造成的上升或下降, 可被GSH reverse 回來), 機制是什麼? 

5. To confirm or extend more experiments, let's try on OKF4-hTert, NP460hTert and CGHNK2.

---

04/14/2015 

1. Focus on nucleosome downregulation: histone cluster 1 on ch6 and histone cluster 2 on ch1.

2. Large hypo-methylation blocks: 在正常細胞時多為LAD/LOCK domain, 經過"transformation stimuli"後, 成為hypo-methyl region.

    LAD: lamina-associated domain

    LOCK: laroge organized chromatin lysine-modification

3. 40T/N methylation array中, delta beta >= 0.35 定義為 hypermethylated genes, delta beta <= -0.3 定義為 hypomethylated genes.

4. 40T/N methylation array中: 

    N_MAX-N_MIN  delta beta >= 0.3 計有6830 (表示 normal tissue中, methylation差異性的存在)

    T_MAX-T_MIN   delta beta >= 0.3 計有10808 (表示 tumor tissue中, methylation差異性的存在)
    T AVB-N AVB  delta beta >= 0.3 計有702 (Illumina廠商根據其它條件 例如是否有CpG island, 再縮減成37個up, 37個down.

5/26/2015 ^New

On Molas portal:

step 1: OKB2 > 1, OKB2_AC > 0,   Fold >= 2, 得 3220 transcripts.

step 2: OKB2 > 0, OKB2_AC >= 2, Fold >= 2 , 得1460 transcripts.

step 3: combine step 1 and step 2, 用 "IF" 公式 , 得 unique transcrpt n = 3563.

step 4: Sort for value, < 0.1者全部replace為0.1, 計算Fold.

step 5: Sort for GeneName / OKB2值大到小 / OKB2_AC 值大到小, 用"IF" 公式過濾unique gene name.

小計: unique gene name n= 2547 (down 1873, up 674).

         unique transcript / no gene name n=155.  

         2547 + 155 = 2702 = 1987 down, 715 up.

---

Among the 2547 unique genes, we will focus on

(1) chromatin organization genes

(2) cell cycle related genes

(3) TP53 related genes (TP53值較高的transcript 因未達2倍差異, 反未被挑出 )

(2) anti-oxidant genes

(3) secreted proteins 

寄送SW780 RNA給長庚及行動基因陳華鍵

寄送SW780 RNA給長庚及行動基因陳華鍵
由 sufang 在 五, 02/13/2015 - 15:20 發表 Cholangiocarcinoma GeneFusion

From: Su­Fang Lin [mailto:sflin1@gmail.com] Sent: Friday, January 30, 2015 2:06 PM
To: angel945@cgmh.org.tw
Subject: this is Su­Fang's email

Dear Dr. 趙:
This is my email. I will prepare ~5­10 micrograms RNAs from SW780 cells for

your control. Please let me know to whom and to where should I sent. Thanks!

Best, Su­Fang 

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

Angel Chao Fri, Jan 30, 2015 at 2:49 PM To: Su­Fang Lin
Cc: 蔡佳龍 , knoxtn@cgmh.org.tw

Dear Prof. Su‐Fang,

Thank you very much indeed for your prompt e‐mail. Dr. Tsai (蔡佳龍博士)will give you the details for RNA transfer to our lab. Thanks again and nice meeting you at this fruitful CGMH‐NHRI meeting.

Regards, Angel

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

蔡佳龍 Fri, Jan 30, 2015 at 4:16 PM To: Angel Chao
Cc: Su­Fang Lin , 王子豪醫師

林老師您好: 非常感謝您願意提供給我們SW780 cell RNA

由於RNA運送須以乾冰低溫運送比較麻煩 不知能否冒昧的跟老師要SW780 cell, 以常溫運送 我們培養後再自行抽取RNA 比較不佔用您的助理太多時間

再次感謝老師的幫忙 敬祝

研安

蔡佳龍敬上 桃園市龜山區復興街五號

林口長庚醫院兒童醫療大樓J棟3樓婦產部實驗室 蔡佳龍
電話:03­3281200­8738或8784

[Quoted text hidden]

Su­Fang Lin Fri, Jan 30, 2015 at 5:10 PM To: 蔡佳龍
Cc: Angel Chao , 王子豪醫師

Dear 佳龍:

因為我們實驗室主要是做頭頸癌, SW780膀胱癌細胞當初是養幾代抽完RNA後就 沒有再maintain, 所以送細胞和送RNA是一樣得用乾冰的. 另外是 SW780我是和人 要來的, 得要再寫信去國外友人處問可不可以分讓,有MTA問題, 反而比較麻煩。

乾冰運送RNA我有經驗、事實上半年前高醫有人跟我要SW780 RNA,目的與你們 一樣,也是當positive control, in which the results looked good. 所以我才有信心 跟趙醫師說沒問題.倒是FGFR3的fusion partner很多、即便同一個partner 斷點 也不同, 因此若欲以RT­PCR 方式篩選的話, 這點要小心.

Best,
Su­Fang
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­-------------------------

蔡佳龍
To: Su­Fang Lin
Cc: Angel Chao , 王子豪醫師

林老師您好: 謝謝的您幫忙與提醒

再麻煩老師了 謝謝您

佳龍敬上 

---------

Su­Fang Lin Tue, Feb 3, 2015 at 2:48 PM To: 蔡佳龍
Cc: 英潔YCK , 玉蓮YLC

Dear 佳龍,
一切順利的話我們應該明天星期三就可寄出冷凍包裹, 我們會請黑貓宅急便送, 標

示貨到付款, 寄到 桃園市龜山區復興街五號

林口長庚醫院兒童醫療大樓J棟3樓婦產部實驗室 這樣子OK嗎? 對了,可能還需要填你們實驗室電話. 謝謝!

Best, Su­Fang 

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

蔡佳龍 Tue, Feb 3, 2015 at 2:55 PM To: Su­Fang Lin
Cc: 王子豪醫師 , 趙安琪醫師

林老師您好: 我們實驗室的電話

03­3281200­8784或8738 我的手機0975510816 再次感謝您的幫忙

佳龍敬上

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

蔡佳龍
To: Su­Fang Lin Cc: 王子豪醫師

Wed, Feb 4, 2015 at 7:27 AM

林老師您好: 我本身是在長庚醫院基因體核心實驗室工作,主要以affymetrix晶片研究基因體

的變化。
目前affymetrix新晶片human transcriptome array (HTA)號稱可以偵測alternative splicing與fusion gene。不知能否跟老師多要些sw780 RNA(10ug)讓我們可以測 試晶片能力,我們也會將晶片資料與老師分享。
再次麻煩老師

謝謝您的幫忙 佳龍敬上

----------

Su­Fang Lin Wed, Feb 4, 2015 at 8:45 AM To: 蔡佳龍 , 王子豪醫師
Bcc: 玉蓮YLC

Dear 佳龍,

其它cell line的RNA可以嗎? 昨天我們有多分出一管OSCC cell line TW2.6的RNA (10 ug 多一點點). TW2.6也有fusion gene, 但不是FGFR3, 而是一個receptor protein tyrosine phosphatase.

因為我們習慣讓分裝好的RNA過夜冷凍, 隔天再寄. 所以要給你們的SW780 RNA昨 天已分裝好冰回. Ten­ug 分一點過去做FGFR3的RT­PCR, 剩下的做HTA應該很足 夠喔! How do you think?

Su­Fang 

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

王子豪 Reply­To: 王子豪

Dear Dr. Lin:

Wed, Feb 4, 2015 at 9:20 AM

Thanks for the reply. If you permit, we will analyze the SW780 RNA with HTA microarrays too and report the results to you as soon as we get them.

Best regards, Tzu­Hao 

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

Su­Fang Lin Wed, Feb 4, 2015 at 9:36 AM To: 王子豪
Cc: 蔡佳龍
Bcc: 英潔YCK , 玉蓮YLC

Dear Dr. Wang,
Are you talking about MTA thing? 據我所知, SW780的 gene fusion至少有兩篇

paper都有發表了, 所以我覺得OK.

上週佳龍問我要cells時,那就會有concern,因為我也是跟友人要來的,要分讓給 你們, 我的習慣是還會請你們跟他簽一次MTA. 想說如果只是做control, 給RNA就 可避掉這些麻煩啊.

Thus, go ahead and use both TW2.6 and SW780 RNA for your HTA analysis. 各 10­ug 應該夠吧, 還是你們需要更多, 那我們就要再回溶一次SW780, 延遲到明天 寄 ­­> Which is also fine with us.

Best, Su­Fang 

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

柯英潔 Wed, Feb 4, 2015 at 4:35 PM To: "hcchen@actgenomics.com"
Cc: 林素芳 , 陳玉蓮

陳博士您好

今天下午已經將RNA樣本用黑貓冷凍寄送給您 預計明天中午前會收到 請依照以下步驟沉澱RNA使用

每管約為12ug RNA溶於formamide,體積20ul,根據以往經驗沉澱後回收約80% 以上,因此最後會得到約10ug的RNA。
沉澱步驟為
1. add 1/10V 3M sodium acetate

2. add 3V EtOH(Merck), mix well 3. ­20°C O/N
4. 14500rpm, 15min
5. 70% EtOH(cold) 100ul wash 6. 14500rpm, 6min

7. Dry pellet
8. Resuspend in RNase­free H2O 9. Nanodrop

 如有任何問題再跟我聯繫
 謝謝

柯英潔 分機35122 林素芳老師實驗室 癌症研究所 國家衛生研究院 

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

Hua Chien Chen (陳華鍵)

To: 柯英潔
Cc: 林素芳 , 陳玉蓮 , "Bernett Hsu (徐安)"

Dear 英潔
謝謝你寄來的 RNA 以及後續處理相關資訊,我會請公司同仁依此實驗流程操

Wed, Feb 4, 2015 at 4:45 PM

作。謝謝!

陳華鍵

[Quoted text hidden]

This e­mail may contain confidential information of ACT Genomics Co., Ltd. If you are not the addressee, please notify the sender and delete this e­mail immediately. Any unauthorized review, use or distribution by anyone other than the intended recipient is strictly prohibited.

柯英潔 Wed, Feb 4, 2015 at 4:48 PM To: "Hua Chien Chen (陳華鍵)"
Cc: 林素芳 , 陳玉蓮 , "Bernett Hsu (徐安)"

陳老師您好

我想再補充二點
1. RNA在formamide中很穩定,如果還沒要往下做,直接丟 ­80C冰箱冷凍起來。 2. 步驟中,­20度沉澱只需要1小時以上即可

柯英潔 分機35122
林素芳老師實驗室
癌症研究所
國家衛生研究院 

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

Hua Chien Chen (陳華鍵)

To: 柯英潔
Cc: 林素芳 , 陳玉蓮 , "Bernett Hsu (徐安)"

Dear 英潔
寄來的 RNA sample 已收到了 謝謝!

陳華鍵 

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

Oligo檔案區

目前想到的方法如下，請大家集思廣益讓「編號引子」成為正確有效又快樂之工作! 謝謝大家~

Step 1: 拉到本文下方第一則回應、看看最新一個人引子的LAB ID是用到第幾號了, 記住你要開始的號碼.

Step 2: 利用本文右下方「增加新的回應」

Step 3: 主題部分標入你login時間及大名 

Step 4: 回去你的Excel檔做編輯

**Excel檔來自生工 : (1) 在最前面加一欄 LAB ID (2) 輸入剛剛記住的號碼並完成編號 (3) copy A to G的資料 paste在ELN回應內文. (4) 記住設定table大小為700 (px) (範例見下面2015/01/01小嬿)

**Excel檔來自每得IDT : (1) 在最前面加一欄 LAB ID (2) 輸入剛剛記住的號碼並完成編號 (3) Move "Tm"、"Final nmoles"、"H₂O to 100 uM"、"product bp" up to "Sequence". copy A to H的資料 paste在ELN回應內文. (4) 記住設定table大小為700 (px) (範例見下面2015/01/23 Ingrid)

建議大家自己有一個Excel檔，專門存放oligo sequences..

ps. 範例2015/01/01 小嬿那一筆的LAB ID是瞎編的，最後會刪掉; 2015/01/23 Ingrid這一筆是真的，請下一個幸運兒從978開始使用! Thanks.

            

2015-02-10 Journal Club Paper

Dear all:

這是我下週要報告的paper

題目為:Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration

敬請多多指教

謝謝

    思涵 敬上

Fusion gene detection 2014-Dec 及 TLCN檢體申請

Fusion gene detection 2014-Dec 及 TLCN檢體申請
由 sufang 在 三, 01/21/2015 - 11:29 發表 Cholangiocarcinoma cholangiocarcinoma FGFR2 gene fusion

Nov 27, 2014 at 2:57 PM

<910208 mail.kmuh.org.tw=""> To: Su-Fang Lin

Dear SF,

Only minor suggestions (in red color). Are you going to list the oncogenes screening in your proposal or we can just perform the exam?

I am current reading most recent driver mutations in the cholabgiocarcinoma, but I will be great appreciated if you can also give me the most updated/recurrent mutated genes in cholangiocarcinoma. Than I will design primers for those candidate genes.

Furthermore, I do not have a MiSeq in my lab yet, so I outsourcing now. I wish

I can get my own MiSeq machine in one year.

Kindly regards, Daw-yang

Thu, Nov 27, 2014 at 4:34 PM

Su-Fang Lin To: Daw-yang Hwang

Three references for you! Best, Su-Fang

4001_2014_03_New Routes to Targeted Therapy of Intrahepatic Cholangiocarcinomas Revealed by Next-Generatiion Sequencing.pdf

4239_2013_12_Exome sequencing identifies distinct mutational patterns in liver fluke-related and non-infection- related bile duct cancers.pdf

3970_2013_12_Exome sequencing identifies frequent inactivating mutations in BAP1, ARID1A and PBRM1 in intrahepatic cholangiocarcinomas.pdf

Daw-yang Hwang <910208 mail.kmuh.org.tw=""> To: Su-Fang Lin

Please see the attached file for the updated TLCN form. Daw-yang

about your application

6 messages

To: "sflin1@gmail.com"

Dear Dr.Lin,

Mon, Dec 1, 2014 at 7:22 PM

Welcome to apply for TLCN samples. However, I need to tell you that many of the cholangiocarcinoma samples are not so good in the RNA quality (due to tumor necrosis), and many have low tumor %. Thus, though we have more than 150 cases, but it is impossible to get 96 cholangiocarcinoma with high tumor % and good RNA quality for your application. we probalbly can give you around 40-50 cases. If you just need DNA and tissue section, it may be possible.

shiufeng

Su-Fang Lin

To: <910208 mail.kmuh.org.tw="">

Dear Daw-Yang,

Mon, Dec 1, 2014 at 9:05 PM

What do you think? Can I tell her that for our purpose not so ok RNA are still worth a try? Thank you very much! SF

Sent from Gmail Mobile

Daw-yang Hwang <910208 mail.kmuh.org.tw=""> To: Su-Fang Lin

Dear SF,

Tue, Dec 2, 2014 at 5:06 AM

I can understand the RNA quality issue. We can try 48 RNA samples first to see how it goes. For DNA, it can be as much as possible, so maybe we should apply 144 DNA samples?

Daw-yang

Su-Fang Lin Tue, Dec 2, 2014 at 9:37 AM To: ,

Dear Dr. Huang,

Thank you very much for telling us the tumor necrosis/poor RNA quality issue. With this regard, we would like to apply 48 RNA samples and associated unstained paraffin sections; and 144 DNA samples of cholangiocarcinoma tissues. A new application form with revised case numbers is attached in the mail.

Thank you very much for your great help!!

Best, Su-Fang

TLCN sample_request_NICR_LinSF v2.pdf 282K

To: Su-Fang Lin ,

144 is too many. Please reduce to 80 to 90 cases.

Do you accept cases with a tumor percentage around 50 percent only? iPhone 

Su-Fang Lin To:  

Tue, Dec 2, 2014 at 12:25 PM

114太多啊! 真對不起，因為機器一次跑48個所以想說取它的倍數來申請...

好，那我們就申請九十例DNA檢體。另外，50% tumor cellularity 應該是OK的。謝謝妳!

素芳

---

Re: about your application

9 messages

 Sat, Dec 6, 2014 at 9:10 PM 

To: Su-Fang Lin Cc:

Please remember to submit a revised form of LOI.

Su-Fang Lin Sat, Dec 6, 2014 at 10:19 PM
To: ,
Dear Dr. Huang,
Enclosed please find my revised LOI. 

Again, many thanks for all your advice and help!

Best, Su-Fang

Letter of Intent of TLCN_NICR_SuFangLin v3.pdf


To: Su-Fang Lin Sun, Dec 7, 2014 at 8:17 AM

You did not change the case number for DNA. Many ICC tumors have low DNA tumor %, and need to use laser capture microdissection, which will be quite expenstive (1000/ 1ug). Anyway, the total number still can not reach 144 cases. Could you reduced to 96 cases (48 wells x2) for DNA?

shiufeng 

Su-Fang Lin To:  Sun, Dec 7, 2014 at 10:00 AM

咦，我在昨天LOI (v3)有改成90例DNA檢體及50% tumor cellularity. 是還有地方漏掉、沒改到的嗎、還是檔案沒有附檔成功? 讓我再送一次! 麻煩您再檢查一下，不好意思。

Best,

Su-Fang

sfhuang@nhri.org.tw To: Su-Fang Lin Sun, Dec 7, 2014 at 8:42 PM

You only made change in the Item V, but you did not change in Item III, No.2, which is the most important part. I can not revised the LOI for you, since it is in pdf file.

shiufeng

Su-Fang Lin Sun, Dec 7, 2014 at 9:03 PM To:

真是糊塗，不好意思. Best, Su-Fang

sfhuang@nhri.org.tw  Sun, Dec 7, 2014 at 9:11 PM

To: Su-Fang Lin

OK. It will be sent out for review.

Shiufeng

Su-Fang Lin To:

Great, thank you very much, Dr. Huang!

sfhuang@nhri.org.tw To: Su-Fang Lin Sun, Dec 7, 2014 at 9:19 PM

you are welcome.

Shiufeng

#1由 sufang 在 三, 01/21/2015 - 15:23 發表。
Regarding sample request at TLCN

Collaborative Projects between KMUH and NICR

General

Fluidigm Access Array + MySeq

–YouTube 1: https://www.youtube.com/watch?v=s9HUhuCbbhU

–YouTube 2: https://www.youtube.com/watch?v=WXpNjcRwdj8

–AA_Illumina_ug_100-3770 (pdf)

–RecoverAll Total Nucleic Acid Isolation Kit for FFPE (pdf)

  台大郭頌鑫醫師抽取DNA and RNA from tiny endoscopic biopsy samples使用。

–陳華鍵老師他們新開的公司行動基因,用的是類似方法、只是不同機器(Ion Torrent PGM/Proton)

–Archer: Archer™ FusionPlex™ FGFR Panel (USD 495 / 8 rxn)

–Life: Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel

–與台大合作部分，詳見HPV and p16 in HNSCC. (按我連結) 

–ds-cDNA protocal from Yi-Mi and Dan (03/05/2015). (按我下載)

–ds-cDNA protocal from 黃醫師Lab 智娟 (2015/04/19). (按我下載)

Gene Fusion Panel

–16個receptor tyrosine kinases (pdf 3815)+ RSPO2/3 (pdf 3594) 

ALK, AXL, FGFR1, FGFR2, FGFR3, NTRK1, NTRK3, PDGFRA, RET, ROS1, MUSK, EGFR,INSR,INSRR, MET, NTRK2, RSPO2, RSPO3

–黃醫師 final list on 2015/04/10

ALK, ABL1, FGFR1, FGFR2, FGFR3, NTRK1, NTRK3, PDGFRA, PDGFRB, RET, ROS1, FLT3

(4 x 96 primers)

Cholangiocarcinoma Panel

ARAF, ARID1A, ARID2, AXIN1, BAP1, BRAF, CDH1, CDKN2A, CTNNB1, FBXW7, FLT4, IDH1, IDH2, KRAS, MLL3, NRAS, PBRM1, PI3K3CA, PTEN, SMAD4, TERT, TP53, VEGFC, VEGFR3, 

hTERTp -124, and hTERTp -126 (526 amplicons)

2015/04/20 Line messages from Dr. Huang: (1) 50 ng for one panel, 2 panels for each TLCN sample. (2) 2 PCRs to increase specificity (3)  if possible, ship 500 ng / 10 ul / sample will be the best. 

TK Panel

Dear Prof Lou,

We will order your primer tomorrow. Beside those oral pharyngeal CA panel, we already have some TK panel, which include: FGFR1, FGFR2, FGFR3, ALK, ROS1, NTRK1, NTRK3, RET, PDGFRA, PDGFRB, FLT3. More TK genes will be added later. We can run your samples in your cancer panel and current TK panel if you are interested. (2015/04/29 email)

sgRNA construction

關於CRISPR sgRNA的設計跟構築

除了委託廠商代工外

現在常用的方法

是利用外切 restriction enzyme去製造linear plasmid

再利用合成stick end 互補股引子的方式去 ligation


常用的restriction enzyme會是類似BbsI這類特別的酵素

因為他們的切點在辨識序列的外側 (5' or 3')來行成stick end

所以只要在合成primer的時後先預留正確的extra base

將回溶/reannealing/PNK 處理過的片段作ligation

就能得到sgRNA vector，

要在經定序後確認，即可得到sgRNA。


目前sgRNA的確認只能用定序的，因為塞入的片段太小(23bp)，

加上sgRNA設計的時候沒有在原始被移除的序列中加上vector其他部位已經有的切位

所以需要這部，或是可以在製造linear vector的時後check 不會有 uncut vector存在


addgene提供的 空sgRNA vector 多數是用這個方法(Humanized sgRNA是例外，我被暗算過)
empty sgRNAhttp://www.addgene.org/crispr/empty-grna-vectors/

裡面也有提供類似冷泉港的單一vector。


利用這個方式建構的sgRNA成本會遠低於委託廠商代工

主要花的錢包含vector購買(NT 3000)/ primer 合成 (~50 bp per clone)/
PNK 跟 restriction enzyme  (各約莫三千元左右，可多次使用)



以下在附上我常用的sgRNA設計網站
sgRNA design
http://www.e-crisp.org/E-CRISP/


最後附上 sgRNA構築的protocol

sgRNA construct

二期癌症缺口計畫2015

2015.01.15(W4) 11:00 am­ 口腔癌缺口計畫會議

總計畫中文: 針對台灣口腔癌前病變惡性轉化開發快速診斷與有效化學預防之整合研究

總計畫英文: Rapid diagnosis and effective chemoprevention for malignant transformation of oral potentially malignant disorders in Taiwan  

  

計畫編號: MOHW103-TDU-212-114005

三月中旬舉行第二次meeting, 討論期中報告繳交．

#1由 sufang 在 五, 05/29/2015 - 13:22 發表。

100例病人葉酸濃度紀錄 謝謝高醫、謝謝奕淇

2015/05 (n=20)

#2由 mmiiee 在 二, 04/14/2015 - 09:09 發表。 口腔癌缺口計劃2015/4/8會議記錄 口腔癌缺口計劃2015/4/8會議記錄 日期: 2015年4月8日 (星期三) 時間: 10:00-12:30 am 地點: 竹南院區 研究大樓2樓 R2-2031會議室 出席人員: (出席人員以螢光筆標示之) 江士昇 助研究員、李岳倫 助研究員、林佩瑩 副管理師、林素芳 副研究員、 夏興國 助研究員、張書銘 副研究員、郭靜娟 副研究員、陳奕淇 計劃助理、 陳雅雯 助研究員、陳嬿如 博士後研究員、張憶壽 研究員、劉柯俊 副研究員、 劉滄梧 副所長 (依姓名筆畫排序)

癌症個人化醫療前瞻論壇 @ 台北喜來登 星月廳 (B2F)

20150111_癌症個人化醫療前瞻論壇 @ 台北喜來登 星月廳 (B2F)
由 sufang 在 日, 01/11/2015 - 20:41 發表 Pre-published Meeting Oral Cancer

Opening remarks: 賴瓊慧教授 (副研發長) 主持人: 王子豪醫師 (國外廠商contact him about STIP1)

閻紫宸教授: 台灣口腔癌病患新穎癒後生物標誌的發現與臨床意義

–Why OSCC: Taiwan has the oral cancer incidence in men worldwide

–Oral cavity cancer: incidence, survival and treatment

–廖俊達教授is the head of CGH HNSCC group leader (pastic surgeon is very important)

–OSCC 2-yr survival: CGMHLK vs all centers

–Clinical path of OSCC

    –全世界一樣的biologic phenotype: ECS positive (survival rate 30%, how to pick them up? )and LN meta

–Unanswered questions: (a) how unique is Taiwan's OSCC? (b) Which genetic alterations can be used as the molecular markers for disease recurrence, metastasiss survival and second primary timor? (c) which targeted therapy can be used for OSCC patients?

–2000年以後是 molecular era, 2010 以後 是 immunotherapy era

–OSCC --LN meta 40% (positive) --? ECS positive (60%) --> 24%

–OSCC --LN meta 40% (positive) --? ECS negative (40%) --> 16%

–OSCC --LN meta 60% (negative) –Comparison 印度 （檳榔子 and no alcohol, 我們的是檳榔汁

–Outline of the study

Stage I: exploration (345 FFPE: ABI 45 core genes targeted based as deep as possible, at least 5-yr follow-up on 345 FFPE samples ; 其實發現妥善處理 應該到1996年的檢體都可用)

Stage II: verification

ultra-deep sequencing of all coding exns of 409 cancer genes using an ndependent cohort (currently, n=300

加入 miRNAs

conclusion: (1)80% found non-synmous mutations and 80% of them are on the top 

potential targetable genes

Stage II 90% found non-synmous mutations NOTCH1增加了

TP53 and NOTCH 與 TCGA相當

Pathwayss affected in OSCC (II)

--110 人 as base , 並以 image (free software abailable)DNA repair 

 

 

tage III: –compare to TCGA data, PI3KCA and TP53 是相當相似的 Notch1 似乎過低 Genetic alterations on prognosis: after 2004 we improve OSCC survival by "team combined care" 

–2015年: proton therapy 10 genes with mutation frequency > 2% were used to construct the OSCC prognostic gene panel with clinical outcomes --> 可以清清楚楚地把345個病人 (1996-2012 ) 分成兩群

–Internal validatio of OSCC prognostic gene panel: either by years (2004 as cutoff) or randomization

Q0: treated 病人還會有多少人嚼檳榔

Q1: CDKN2A (p16)  婁培人醫師: Taiwan p16 status: more mutations than what have been reporter in the Caucasians. (存疑)

                                廖仲達醫師: E6 E7 expressions are related to metastasis of oral cancer. 已發表蠻多篇相關papers

Q2: validation by TCGA data or Indian data 每一年3% 的survival patients 又會長出OSCC related: field cancerization NGS gene panel for developing primary tumor Stage 3: prognostic markers? 409 cancer genes in important signaling pathways cover 10 cancer genes and more than wp essential cancer pathways 

---

胡松年博士: 消化性結腸炎癌病患者的基因變異圖譜分析

003-2011年來台多次 UC的病人後來成CRC的機率很高

whole genome : HiSeq 2000 and ion proton

數據庫: GWAS, CLINVAER, COSMIC, HGMD_

1 case clinical sample Exom-seq 100X germ-line 突變很高 -> DNA repair發生問題? 但四個數據庫裡頭都有的只有0個; 後改成任兩個有的則多落在germ-line突變

IBD 與 CRC 都有的 germlline mutations are similar to that reported by others DNA repair point mutations

CRC mutational landscape 1000 Genome

CHS (南方漢族人) vs CHB (北京漢族人)

208 WES and low coverage WGS --低頻mutation in DNA repair system -- 7 DNA repair system 均有 -- 106 genes

---

陳淑貞: personalized medicine

​

–Cancer is a disease of genetic alteration

–Genetics for precision cancer medicine (diagnosis, therapeutics and prognosis)

–NGS and clinical oncology (NGS technology advancement; NGS for clinical oncology) 2013 FDA approves 2 important platform and 2014 FDA improves another platform.

–High accuracy error rate ~0.1%;high sensitivity : 1-3%; high specificity; cane detece all tpes of genetic relationship associated with cancers

–Eric Green 2011 年 presented: from base pairs to bedside Nature 2011 (pdf 2868) (ENCODE, TCGA, 

NCI+NHGRI initiated on 2006 (3-types) --> 2009 (20 cancer types)

ICGC project maps 

Pan-cancer analysis  (pdf 4139, 4140)

-A CUP patient harbors 16 copies of MET -? crizotinib cure for >19 month

-Genetically matched therapy are more effective (on TN-breast cancer)

-based on these results NCI-MATCH (plan to perform on 180 patients, molecular analysis for therapy choice trial)

-Outlier patient obtained better treatment than other patients 

1. 2012 Science: Everolimus sensitivity marker on bladder cancer: NF2 TSC1 "exome-sequencing of ...)

2. Cancer Discovery: Metastatic urothelial carcinoma : mTOR everolimus sensitivity 

3. NEJM: Anaplastic thyroid cancer : everolimus  TSC2 mutation -> 14-month

 

Cetuximab resistance in mCRC patients

-32 (responders): 20 (non-responder) Genetic alterations asspciated awith Cetuximan resistance 

–只sequence EGFR-RAS-MAPK 

–Non-responder K-RAS exon 2 在TCGA data裡頭已被filter掉

—NRAS 

—BRAF V600E is druggable

—ACT genomics: turn genomics into action

Start: 01/11/2015

2014-01-20 冷泉港 CRISPR 說明會

大家好，

謹訂 1/20 下午1：30 在 R1-1041 舉辦 CRISPR 說明會，

由冷泉港的技術專員 陳博士與李小姐 負責解說，

內容包括 CRISPR 原理與應用、冷泉港 CRISPR 產品介紹與操作方式，

還有示範如何設計 guide RNA

如有其他問題或需求，歡迎現場提問，

請大家務必準時到場，謝謝

另外，當日的 Journal Club 順延一次

Nature. 2014 Dec 18;516(7531):423-7. doi: 10.1038/nature13902. Epub 2014 Oct 22.

In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system.

Maddalo D1, Manchado E1, Concepcion CP2, Bonetti C1, Vidigal JA1, Han YC1, Ogrodowski P1, Crippa A3, Rekhtman N4, de Stanchina E5, Lowe SW6, Ventura A1.

Author information

Abstract

Chromosomal rearrangements have a central role in the pathogenesis of human cancers and often result in the expression of therapeutically actionable gene fusions. A recently discovered example is a fusion between the genes echinoderm microtubule-associated protein like 4 (EML4) and anaplastic lymphoma kinase (ALK), generated by an inversion on the short arm of chromosome 2: inv(2)(p21p23). The EML4-ALK oncogene is detected in a subset of human non-small cell lung cancers (NSCLC) and is clinically relevant because it confers sensitivity to ALK inhibitors. Despite their importance, modelling such genetic events in mice has proven challenging and requires complex manipulation of the germ line. Here we describe an efficient method to induce specific chromosomal rearrangements in vivo using viral-mediated delivery of the CRISPR/Cas9 system to somatic cells of adult animals. We apply it to generate a mouse model of Eml4-Alk-driven lung cancer. The resulting tumours invariably harbour the Eml4-Alk inversion, express the Eml4-Alk fusion gene, display histopathological and molecular features typical of ALK(+) human NSCLCs, and respond to treatment with ALK inhibitors. The general strategy described here substantially expands our ability to model human cancers in mice and potentially in other organisms.

PMID: 25337876

帥呆了! 要來做做看嗎?