Saturday, December 3, 2011

Open Science Forum: DIPG Preclinical Consortium

Check back frequently to this blog post, which gives a week to week account of the project entitled, "Rapid Preclinical Development of a Targeted Therapy Combination for DIPG" described here and here. This study is made possible by a $100,000 grant from TheCureStartsNow. An additional $28,000 supplement from The Lyla Nsouli Foundation for Children’s Brain Cancer Research has allowed our project to expand to our 2 participating European collaborators.


The full international team is:


Charles Keller MD, Kellie Nazemi MD, Nate Selden MD, PhD and Dan Guillaume MD, PhD at the Oregon Health & Science University
(cell lines:  OHSU-DIPG#1 now senescent, OHSU-DIPG#2 now senescent
Oren Becher MD, Duke University Medical Center
(cell line, from mouse:  11.1003.2, which is GFAP tva:floxed PDGF p53) 
Michelle Monje MD, PhD, Stanford University
(cell lines:  SU-DIPG-I, SU-DIPG-II, SU-DIPG-III, SU-DIPG-IV, SU-DIPG-V, SU-DIPG-VI)
Maryam Fouladi MD, Cincinnati Children’s Hospital Medical Center
Cynthia Hawkins, MD, PhD, University of Toronto
(cell lines: HSC-DIPG27, HSC-DIPG58)
 Xiao-Nan Li MD, PhD, Baylor College of Medicine
(cell lines:  IBs-1215DIPG, IBs-A1204DIPG, IBs-AB10206DIPG, IBs-C1220DIPG, IBs-J1204DIPG, IBs-W0128DIPG)
Dannis G. van Vuurden MD, MSc, & Esther Hulleman, VU Cancer Center Amsterdam, 
(cell lines:  VUMC-DIPG-B, VU-10170)
Jacques Grill, Institut Gustave-Roussy, Villejuif, France
(Nem144, Nem145, Nem157, Nem163, Nem165, Nem168, Nem175, Nem181, Nem186 and Nem198; Nem215 and Nem226
and

Eric H. Raabe, Johns Hopkins University, Baltimore, MD; Katherine Warren, Paul Meltzer and Martha Quezado, NIH, Bethesda, MD
(cell line: JHH-DIPG1)
Mariko DeWire, Nationwide Children's Hospital, Columbus, OH
(cell line name TBA)
Dennis Steindler, University of Florida, Gainesville, FL
(hDIPG-L1)
Marta Alonso, University of Navarra, Madrid, Spain
(tba)

The ultimate goal is to move the most effective single agent or combination therapy forward to early phase clinical trials in the next 18-24 months. The clinical trial would be available from the Children's Oncology Group, and/or collaborating institutions. This is the first time that a group of basic and translational scientists and physicians from eight institutions throughout North America and Europe have come together as a consortium to focus on DIPGs and to focus on a bench-to-bedside approach to rationally target therapy for children with DIPGs.


Project Progress:
11/01/2011: The CureStartsNow and affiliated foundations provide $100,000 for project for labs in North America.
11/21/2011: Prototype drug plates sent to Becher, Li and Monje labs (shipping to Hawkins lab tba).
11/23/2011: The Lyla Nsouli Foundation provides $28,000 to add participating European labs.
11/23/2011: Prototype drug plates sent to Grill and Hulleman labs.
11/28/2011: Concept of exon sequencing of paired cell lines, primary tumors and normal DNA (via tertiary funding source) considered.
12/08/2011: Subcontract paperwork to each site has been initiated.
12/08/2011: Plastic tips for robotic drug plating on order (needed to create more plates).
12/08/2011: (Keller lab) Plan initiated for biomarker characterization of OHSU-DIPG#1 cell culture.
12/08/2011: (Keller lab) Sequenom MassArray screen completed for OHSU-DIPG#1 cell culture.
12/15/2011:  Plastic tips for drug plating were backorder from Eppendorf but may arrive in the coming days.  Plan to create/send 384-well drug plates to Cynthia Hawkins group once tips are in. Also sending additional plates to Oren Becher lab at that time.
12/15/2011:  After extensive review, a generation #2 drug plate set has been designed.  60 agents across 3 plates, dozens of molecular targets.  Incorporates the consensus preferred compounds from labs of Jim Olson, Maryam Fouladi, Robin Jones, Darren Hargrave, Jacques Grill and Esther Hulleman.
12/21/2011:  Plastic tips were received this week and new drug plates have been printed and will ship to the Li lab and Hawkins lab soon.  Experience with the plates thus far (Becher lab) suggests that a new plate types (non-clear/black plastic) with or without gelatin undercoating may be needed.  Assessment will be made once each of the other labs has experience with the generation #1 plates.
12/23/2011:  In an amazing development, ABC2 and CureSearch for Children's Cancer have jointly funded a supplement to the Preclinical Consortium project (with $76,000 and $25,000, respectively).  This new supplement will allow us to exon sequencing of all 16 cell lines, their primary tumor and paired normal DNA via the functional genomics laboratory of Dr. Paul Spellman (OHSU). From this data we will prioritize combination drug testing on the most clinically ‘representative’ samples (i.e., samples that have classical DIPG findings which are in order: c‐MET amplification, p53 deletion, PDGFRA amplification, IGF1R/IGF2 amplification, CDK4/CDK6 gain or Shh pathway mutation, EGFR amplification or KRAS/NRAS mutation) or cell lines that are most similar overall to one another (i.e., not outliers).
01/05/2012:  Each lab was requested to organize samples for DNA sequencing; correspondence from Stanford and Amsterdam was received; further more active correspondence expected as the new year ensues.
01/12/2012:  Most of the cell cultures from participating centers are now verified by name, and in some cases new cell cultures are available.  
01/12/2012:  The first drug screen on a generation#1 drug plate set has been performed by the Becher lab using a mouse DIPG culture.  A logistical success, and at least 8 interesting cytocidal drugs have been identified. 
01/19/2012:  Our first probabilistic boolean model (PBM) of the data above is from the mouse DIPG cells is being generated by our collaborators at Texas Tech, Dr. Ranadip Pal and graduate student Noah Berlow.  
01/26/2012:  PBM data still pending (results soon); DNA or RNA samples from DIPG cultures yet to be received from each institution (for sequencing; target date to receive all samples is Feb 9); 24 new sets of generation#1 plates printed for sending out tomorrow to Toronto (for use with potential new primary culture) and other sites; interesting spherical cell culture beads with different coatings being explored at OHSU as a potential way to accelerate DIPG cell culture growth ;official press release now available here
01/28/2012:  First (draft) PBM tumor maintenance circuit now created for mouse DIPG cells.  Thank you, Noah!  Many known targets were affirmed, but new ones were found, and co-dependencies were identified.  Validation experiments will follow. 
02/02/2012:  New generation#1 drug plates sent to Toronto and Duke; orders submitted for 55 of the 60 drugs to be used to create generation#2 drug plates; DNA/RNA samples received from Stanford and Toronto.  Many public questions asked about the bioinformatics approach.  For clarification of PBM tumor circuits a movie is available here.  
02/09/2012:  samples for deep sequencing received from Amsterdam, Stanford and Toronto.  Additional samples from Stanford and Baylor to arrive today.  Second run of raw data for mouse DIPG culture drug plates  received from Duke, with analysis now underway.  
02/16/2012:  Second run of drug screen from Duke mouse model consistent with first run; Partial data for 30 drugs screened received for SU-DIPG-II from Stanford.  One strong drug 'hit' found in common with Duke mouse model; DNA and RNA sequencing of samples is to start shortly.  
IMPORTANT UPDATE:  Another cell line has been added as an with an new affiliate investigator & participating institution.  A DIPG primary cell culture being studied at Nationwide Children's Hospital in Columbus, OH, by Dr. Mariko DeWire.  Characterization will include focused genetic characterization and drug screening.  This additional affiliate is being made possible by the generous $8800 sponsorship of the Team Julian Foundation.  
02/23/2012:  DNA and RNA for sequencing can be summarized as:  we have samples related to 19 independent cultures:
  
DNA from the primary tumor from 5; 
RNA from the primary tumor from 0;
DNA from normal tissue from 12;
RNA from normal tissue from 5;
DNA from xenograft tissue for 7;
RNA from xenograft tissue for 0;
DNA from tumor cell cultures from 12;
RNA from tumor cell cultures from 8.
  
Complete sets of DNA & RNA from tumor cultures, the original primary tumor and normal tissue were not available from all samples; however, we are moving forward in the interest of time (and the goal of getting the clinical trial designed in the near term).
02/23/2012:  The results for the second half of the first SU-DIPG-I drug screen are received and are being processed.  Many thanks to Michelle (and Oren) for being among the best and most proactive members of the consortium early on.  
03/01/2012:  Common target(s) found in human SU-DIPG-I cell line and mouse 11.1003.2 cell line drug screens; preliminary results to be presented Monday March 5 at PBTC meeting.  Results of SU-DIPG-II cell line now being analyzed.
03/05/2012:  Organization and early results of the Consortium presented at the Pediatric Brain Tumor Consortium (PBTC) closed meeting in Dallas.  Very well received.  
03/08/2012:  In processing RNA and DNA for sequencing, some samples were not of sufficient quantity.  We have contacted each collaborator to send new aliquots for these samples. 
03/14/2012:  Please see our guest blog entry from Nettie here.  
03/15/2012:  The DNA and RNA sequencing continues to be delayed.  Each lab is actively creating supplemental DNA or RNA so that we can begin sequencing April 1 (our new deadline). 
03/22/2012:  there is nothing new to report this week (sadly).  We are just waiting for the replacement DNA's and RNA's to be sent in from the consortium members.  
03/29/2012:  Preliminary results (similar to those presented at the PBTC meeting) were presented at the COG Brain Tumor committee meeting in Minneapolis.  Discussions were active to extend the same approach to other pediatric brain tumors, including high risk medulloblastoma, high grade gliomas, and intracranial germ cell tumors.  
04/02/2012:  At AACR a key new presentation from the Raabe lab was today, "High-level activation of the Notch pathway in diffuse intrinsic pontine glioma" by Hutt et al (a Johns Hopkins - NCI collaboration that generated a new cell line/mouse model of DIPG).  Also presented by Dr. Ian Pollack are the promising early results of a DIPG vaccine trial (caveats not withstanding).  Photo to right is Dr. Eric Raabe and staff scientist, Marianne Hutt.  
04/05/2012:  subcontract financial paperwork now in place (completed) for Institut de Cancérologie Gustave Roussy.  
04/12/2012 (late entry):  DNA samples still be processed for sequencing.
04/19/2012:  Most samples submitted pass quality control to move on to DNA exon sequencing (see table to right).  Only 2 samples overall failed, and these were the normal tissue controls.  We are thus moving on to exon sequencing.  However, our OHSU sequencing core has an 8-10 week backlog.  Therefore we are looking at sending out the samples for sequencing elsewhere.  In addition, RNA from the samples have not been fully processed/analyzed because the manufacturer of a key reagent are backlogged at least several weeks.  There appears to be no way around this reagent backlog.  Meanwhile, "version 2.1" drug plates are nearing ready for printing on the robot.  We have 5 of the 60 ordered drugs yet to receive before we start printing.
ps.  a special thanks to Keith Desserich for organizing yesterday's DIPG Collaborative webinar.  
04/26/2012:  Manufacturer kits to pre-process RNA for sequencing came in this week, and the samples will be prepared next week.  We are weighing options of sending DNA samples for sequencing at OHSU (a longer wait but cost will decrease in that period) versus a secondary service provider outside of OHSU.  We also have an informal participant in the Raabe laboratory, who received v1.0 drug plates for their new DIPG primary cell culture.  This participant and the related expenses are unfunded, and we would welcome a community gift to bring this work more formally into the Consortium.
nb.  Another new DIPG primary cell culture has been established at OHSU, but not in time for sequencing.  Thanks as always to Michelle Monje for the culturing advice that makes this possible for OHSU and other institutions to successfully grow this type of tumor cells.  
05/05/2012:  DNA samples now submitted for sequencing at OHSU core facility.  If we were a regular customer, it might take 2 months.  We've been moved up the queue somewhat with the understanding that our project is time-sensitive.  RNA sample pre-processing is still not performed, but might be in the next 1-2 weeks, with sequencing results likely to follow in another ~ 2 weeks.      
05/10/12:  DNA sequencing *might* get run in the next week if we are lucky.  There is no change in status of RNA sequencing.  No progress on drug testing pilot from Toronto thus far.  Amsterdam labs are working out how to evenly plate spheres into drug plates (this is an issue for all labs; breaking apart cells leads to poor survival, but quantifying cells in an intact sphere for even plating is not easy).  Villejuif lab has several cell lines, but has requested more plates.  The prior shipment of plates was held in customs and thawed out too long, which may have impacted the usefulness of the first set of plates that were sent. 
05/17/2012:  RNA processing for sequencing to be done in the coming week.  DNA final quality check shows that 3 formalin-fixed, paraffin-embedded samples failed (38 of 41 samples passed).  Raw DNA sequencing data may be available as early as 5/27.   Dr. Monje sent (yet) another set of drug screen results from a human DIPG culture (SU-DIPG-VI)... overall similar to past results with other cultures, which is good to see. Also, Dr. Kristin Schroeder's abstract from the Becher lab has been accepted for an oral presentation at the ISPNO meeting in Toronto next month.  Finally, we may have (accidentally) identified a way to improve propagation of early DIPG cultures.  Validation experiments are ongoing.       
05/24/2012:  No new news about DNA or RNA sequencing.  The lab of Esther Hulleman succeeded in screening a new DIPG cell line, VU-10170.  Results were similar to all other screens, with a special sensitivity only seen in one other cell line.  The lab of Jacques Grille is making a strong late-breaking showing in a cadre of 10 DIPG primary cell cultures for DNA and RNA sequencing, as well as drug testing.  Brian Rivera from Eppendorf made a service visit recently to improve the efficiency of our robot, which will be very helpful in creating version 2.1 drug plates (to be distributed after analysis of the main batch of DNA and RNA sequencing is analyzed by Catie Grasso in the OHSU Spellman lab. Another good week. 
05/31/2012:  The first (large) batch of DNA sequencing data is done!  Analysis is ongoing by Catie Grasso in the Spellman lab.  No new news on the RNA sequencing samples.  We're waiting for a new large batch of DNA's and RNA's from the Grille lab, as well as the ok to ship additional drug plates to them.  Special note:  Maryam Fouladi as new head of the PBTC is organizing High Risk Medulloblastoma and High Grade Glioma preclinical consortia based on the COG Preclinical Consortium for DIPG.  These other initiatives are also very worthwhile, but have no funding (and cannot move forward without it).  Please spread the word! 
06/07/2012:  The only new news is that the set of 10 drug plates safely arrived in the Grille laboratory this week, and that replacement DNA's have also arrived from the Li lab.  
06/14/2012:  no new news.  I'm sorry.  The batch#1 genomic DNA is still actively being analyzed, and the batch#1 RNA is still in the queue for sequencing.  No new drug testing results.  
06/21/2012:  no report on account of being out of office. 
06/28/2012:  Genomic DNA still in analysis.  RNA libraries (prepared material) will be sent to the OHSU core facility for sequencing next week.  Grille laboratory DNA and RNA samples (many new ones) still expectant to arrive.  
07/05/2012:  new drug screen data now available from Grille laboratory for Nem145.  This is a total of 7 cell lines that have been drug screened (one mouse, 6 human).  Partial DNA sequencing analysis is ongoing.  DNA sequencing is partially complete (click on updated table to right).  We are waiting for the core facility to have enough samples to go with our samples for a full sequencing "run".  We are conscious of the need to move to combination testing and in vivo testing very soon if we are to create the drug combination clinical trial that was the main purpose of this preclinical consortium.  Thankfully, too, a leader from the community has offered to fund the participation of  the Raabe lab to incorporate cell line JHH-DIPG1 into our studies.  More officially on that very important grant soon.  
07/13/2012:  RNA samples are at the sequencing core awaiting run when space on the instrument is available.  Interim drug screen data is in the hands of clinical trial leaders. Drug screen 2.0 plates may be printed in the next 14 days.  Nothing is more important than to integrate existing data and move to experiments in mice before the September COG meeting.
07/20/2012:  Li lab drug screen performed, consistent with other 7 cell line screens.  Heuristic combinations being tested ad hoc per request of Cincinnati collaborator.  Project is being accelerated towards September COG meeting date.
07/20/2012:  No news on sequencing.  Balance between immediacy of data for planning trials at cooperative groups is intersecting with due diligence of scientific process (delicate balance).    Note, too, the recent thoughtful thesis publication of Viola Caretti, Pioneering Preclinical Research in Diffuse Intrinsic Pontine Glioma: Towards New Treatment Strategies" (ISBN 978-94-6191-323-4, publisher www.ipskampdrukkers.nl).  
08/02/2012:  No news on sequencing.  In anticipation of 7 DIPG parents being at the Pediatric Cancer Nanocourse next week, we're creating an action plan for a Sept 10 "best available" analysis to present to the COG brain tumor cmte.
08/09/2012:  4 new DNA samples have been sequenced.  excitingly, Nathalene Truffaux from the Grille lab just shipped 20 new DNA and RNA samples from DIPG primary cell cultures (DHL en route).  In addition, an action plan is in place, catalyzed by our Nanocourse attendees (picture above).  Under the leadership of Paul Spellman and Catie Grasso, sequencing will be accelerated and analysis performed for drug-responsive DNA and RNA biomarkers by September 10.  Our thanks to these amazing genomic biology scientists! 
08/16/2012:  The Pediatric Cancer Nanocourse parent alumni (now faculty) are finalizing the manuscript on overcoming barriers to cure for DIPG.  For the consortium project, Grille lab DNA and RNA samples are being pre-processed.  Data from two Baylor DIPG culture drug screens are being repeated for thoroughness.  Version 2.0 drug screen plates began being printed today.  Validation is forthcoming.   
08/23/2012:  Version 2.0 plates have low error/high reproducibility. Time pressure exists to get results to COG and PBTC for a feasible drug combination based on v1 and v2 plate results.
08/23/2012:  Catie in the Spellman is making great strides to get the following questions answered before the COG meeting on Sept 11:

-  of the DIPG cell lines studied, samples are outliers? (vs which are more similar)
- are there any driver mutations? (new or known)
- what gene expression or copy number abnormalities correlate with drug sensitivities?


A summary of RNA samples pre-processed (ie, a library was made) and then sequenced is to the right.  Those not yet sequenced in time for the COG meeting will be used as a validation set.  

    

about Catie:
" My career in genomics began in 1999 when I did the data analysis for the first whole human genome annotations of alternative splicing, which is the regulated editing of genes before they are made into proteins.  More recently, my research has focused on cancer genomics:  I just finished leading the largest sequencing effort of metastatic prostate cancer to date, an effort that was recently published in Nature.  We are using the methodology from this study in the context of DIPG in the hopes of making similar breakthroughs." 
    
09/06/2012:  DNA mutations, amplifications and deletions will be known in 1-2 days for a lead set of samples associated with the drug screens.  Xenograft results will follow soon thereafter (requires special care to subtract mouse sequences from human sequences).  Plates for v2.0 drug screen, and a compound related to a v1.0 drug screen lead go out on Monday or Tuesday.   


09/13/2012:  Jacques Grille and his lab team member, Nathalène Truffaux, have contributed 2 additional DIPG cultures:  Nem215 and Nem226.  In addition, Nem175 cell culture drug screen v1.0 results are now available.  Nathalène and Jacques (pictured to the right) have added a great deal of depth to our consortium - we are grateful for their participation;
in addition, interim results were presented at the COG meeting today (including exome sequencing result for 8 tumors)... well received some very good suggestions to incorporate.  Drug screen v2.0 plates delayed in shipping out to other labs until Monday.       
  
09/20/2012:  The COG brain tumor committee presentation of the DIPG preclinical consortium results (including preliminary dna sequencing - drug sensitivity correlations) was well received in Atlanta.  Goal is now to complete dna & rna sequencing and drug plate v2.0 screens before December.   Also, the International DIPG Registry has announced its launch.  
  
09/27/2012:  Seeking additional tumor samples (even without cultures) from collaborators to get better statistical power on the sequencing analysis.  v2.0 drug plates now distributed (to N. American institutions... need to print many more plates for European sites).
  
10/04/2012:  Results from v2.0 drug plate of mouse DIPG primary cultures are very encouraging... new agents on these plates (chosen by the international neuro-oncology community) do appear to have interesting activity against (at least) the mouse dipg cells.
  
10/11/2012:  Results of screens now in from VUMC and Baylor labs (1 dipg culture each, including IBs-1215DIPG).  Paul & Catie continuing to analyze DNA sequencing data.
  
10/18/2012:  ongoing support to Monje lab regarding potential combinations; additional v2.0 drug screen from Li lab (on IBs-W0128DIPG) looks very promising.  Catie still working on DNA and RNA sequencing data as they continue to be generated.

10/25/2012:  Sequencing Facility openings make it possible that 17 additional samples will be run in the next 1-2 weeks.

11/01/2012:  Meeting with bioinformatics team, it appears that about 3 of 7 xenograft samples will have a human:mouse DNA content balance that will allow useful information to be gained.  Met with Sequencing Facility director earlier this week with assurances that our project is felt to be important and prioritized.  (note:  no RNA sequencing data has yet been generated, though it is highly anticipated).  Still gathering important sample characteristics from collaborators with which to make bioinformatics analysis.  Additional v2.0 drug plates go to Grille and Hulleman labs on Monday.  
  
11/08/2012:  More v2.0 plates sent to Hulleman and Grille labs.  RNA sequencing data very close to being performed.  Thanks to Catie Grasso for kindly integrating mixtures of multiple types of data.  ABC2 introduction made to industry-associated community group that could assist with data storage and perhaps analysis.  
  
11/15/2012:  Sequence analysis ongoing.  no other new results.
  
11/22/2012:  Holiday (no progress this week).
  
11/29/2012:  Four additional samples sequenced.  Bioinformatics analysis of probable mutations in DNA sequenced sample is well underway.  
  
12/06/2012:  Preliminary DNA sequencing analysis complete, with confirmation of known mutations and identification of potential new targets. French DNA samples are sequenced, but need to be added to the analysis.

12/13/2012:  DNA sequence analysis ongoing; RNA sequencing done! and analysis also underway.  The Grille laboratory has sent a large number of new version 2.0 drug screen results for integration.  The bioinformatics team from the Spellman (OHSU) and Pal (TTU) laboratories are now working together to integrate sequencing and drug sensitivity results.  This is an exciting week!
  
12/20/2012:  Sequence analysis ongoing; final batch of v2 drug plates need to be printed to send out to 2 participants.  Funds spending down appropriately as grants end.  We're grateful to Catie, Noah and Ranadip for all their hard work on analysis, as well as Eric, Jacques and Nathalene who are working so diligently to get the final experimental data. 
  
12/27/2012:  New batch of v2 drug plates printed (thank you, Mat!).  Will not ship out until after holidays.  No other updates.
  
01/03/2013:  Happy New Year.  getting back on track in preparing shipments of plates. 
  
01/10/2013:  thanks to Catie, the DNA and RNA sequencing analysis is mostly complete.  Exciting expected and unexpected findings.  Version 2 plates shipped to Raabe and Grille labs.  Pal lab is awaiting drug screen results to cross-correlate genomic findings with drug sensitivities.  Chances are good of designing useful 2-drug combination, but we'll see. 
  
* if any foundations are willing, we could use ~ 25k USD to bring together all preclinical consortia investigators for a first face-to-face consortium and data analysis working meeting.
  
01/17/2013:  RNA sequences not previously incorporated bring that count up to almost 30 (controls and replicates are included in this tally).  NEM-215 and NEM-226 still need to be processed & sequenced in the coming few weeks.  JHH-DIPG1 cell pellets received today (thank you, Eric and team) for DNA and RNA sequencing with NEM-215 and NEM-226.  
  
01/24/2013:  Many thanks to Elaine who has compiled the drug screen results to date in a single summary file (including absolute and relative IC50's).  All other analysis are ongoing.  Collaborators have begun adding dipg biology knowledge to Catie's sequence analysis results.  
  
01/31/2013:  NEM-215 drug screen data from Grille lab looks very good.  De-identified tumor features (age, histology, survival) from samples being integrated into bioinformatics analysis. All consortium members working to expedite last final experiments to be completed.
  
02/07/2013:  All final sample experiments and analysis still ongoing.  In addition, we have opened discussions with Dan Silver and his mentor, Dennis Steindler, at the University of Florida, to participate in the consortium.  Our thanks to David Sandak at ABC2 for making this introduction.
  
02/14/2013:  Interesting drug screen results for JHH-DIPG1.  DNA and RNA sequencing pre-processing (libraries) done for JHH-DIPG1, NEM215 and NEM226.  Drug screen plates sent to Steindler lab.  
  
02/21/2013:  Catie will be working on RNA sequencing outlier analysis so that Pal lab (Noah!) can complete probabilistic boolean modeling correlations with the sequencing data.  These are critical elements of the project now.  The last few (straggler) DNA and RNA libraries submitted to sequencing core.  Very constructive comments and new data from Grille lab.  April 10 COG meeting is a hard deadline for manuscript answer and selection of combined targeted agents for next testing with in vivo preclinical testing.  

02/28/2013:  Catie has completed outlier analysis for RNA sequencing.  

03/07/2013:  34 days to go! until COG meeting and presentation of results.  Here is the rundown thus far:
  
- DNA sequencing on tumors, cell lines and/or paired normal samples x 33 independent tumors with 2 more pending.
- RNA sequencing on 25 independent tumors with 2 more pending.
- Drug screens (v1, v2 or v2.1) on 21 samples (5 tumors run more than once on different versions) plus 4 additional drug screens just in from the Grille research group (thank you, Ludivine!)
- Integrate of sequencing and drug sensitivities underway with Ranadip and Noah.
  
Thanks, too, to J.D. and Myron for creating the FTP sharesite and compressing the sequencing data to a size that can easily be exchanged back to the originating labs (respectively).  
  
03/14/2013:  Becher, Monje, Hulleman and Grille labs each working on aspects of project interpretation.  Catie leading manuscript write-up.  Our continued thanks to Paul Spellman for his team's incredible support of the genomics studies (including the tenacity of team member Nick Wang).  
  
03/22/2013:  Final samples for sequencing were submitted 2/19 and 3/21. They are:  
DNA (JHH-DIPG1, NEM-215, NEM-226, OHSU-DIPG#2 Normal, OHSU-DIPG#2  TUMOR) and RNA (JHH-DIPG1, NEM-215, NEM-226).  Ludivine submitted NEM-157 v1 drug screen results.  Noah completed the individualized tumor cell survival circuits (that he calls PTIM's) just today.  Catie and others across the consortium are busy analyzing data and writing up results.  
  
03/28/2013:  Last library samples sent for sequencing.  NEM-157 v1 drug screen were fruitful.  Noah, Catie, Michelle, Oren, Jacques and Michelle have all been very actively working on analysis this week.
 
04/04/2013:  Preparations for COG ongoing, very actively. Catie in full swing, but contributions from Monje, Grille, Hulleman and Becher labs are very strong.
 
04/11/2013:  It all seems worth the work from everyone.  We have a clear direction from the data for 2, if not 3 drugs. The CNS committee gave valuable input, and now we are working towards in vitro (petri dish) validation of drug synergies, then mouse models. Unexpected targets were revealed, and we hope all this work will make a tangible difference. What a great team across so many labs. 
  
04/18/2013:  lab teams in Europe and U.S. working towards the goal of validating drug combinations (in comparison to agents in COG dipg phase I/II pipeline).  contributions and insights of Kathy Warren and Esther Hulleman particularly appreciated.  Monje lab working to get new DIPG-IX drug screen results; Becher lab screening 2-4 additional mouse TVA cultures.  Catie integrating last few DNA and RNA sequencing results.  Michelle Monje to present briefly preliminary studies at The Cure Starts Now meeting next week.  

04/25/2013:  Additional data in process of being generated by Steindler lab, as well as Monje lab.  Sequencing of DNA and/or RNA for last few samples from Grille, Keller and Raabe labs now done.  Catie and others working on manuscript. Dr. William Weiss (UCSF) will join consortium for in vivo testing phase.  
  
05/02/2013:   Michelle Monje presents update of preclinical consortium collaborative work at TheCureStartsNow symposium in Cincinnati, Ohio.  Lots of discussion on how best to present drug screen data (what format) for manuscript.  Brief logistical summary of this COG-related consortium work to be presented by Dr. Oren Becher at Pediatric SNO meeting.  
  
05/09/2013:   Additional Becher lab mouse dipg cell culture cultures examining the impact of histone mutations have been tested on the drug screen.  Experiments still underway in the Steindler lab.  Manuscript writing by Catie in progress.  Jacques Grille will present selected results at the upcoming June 2013 SIOP meeting in Warsaw (our thanks to the SIOP investigators for reaching out to collaborate!)
  
05/16/2013:  New dipg and comparative pediatric HHG culture drug screen results now in. Manuscript writing by Catie in progress. 
  
05/22/2013:  Manuscript writing by Catie in progress. 
  
05/30/2013:  A lot of email traffic preparing figures & tables with collaborators!
  
06/06/2013:  Exploring novel mutations found in dataset.  Interestingly, these are pharmacologically amenable.  
  
06/13/2013:  We gratefully welcome Dr. Marta Alonso from the University Hospital of Navarra to the DIPG Preclinical Consortium.  Marta's role will be in in vivo validation of drug screen combinations, alongside complementary studies in the Monje, Becher and Raabe laboratories.
  
06/20/2013:  Preliminary in vitro drug combination validation data are encouraging.  Hulleman lab to also participate in animal validation studies.  Congratulations to the Lyla Nsouli Foundation on their recent Gala.   
  
06/27/2013:  "Next step" in vivo (mouse) preclinical projects are firmly outlined by consortia members, with funding requests to follow.
  
07/03/2013:  Ongoing manuscript writing in progress.  Additional samples from Grille lab to be sequenced.  
   
07/10/2013:  New experiments explore the transcriptional changes attributable to an effective drug. 
  
07/17/2013:  Manuscript maturing... the goal is to get this data public as early as possible.  Will focus on 2 classes of drugs and 3 interesting gene mutations. 
  
07/23/2013:  Next steps of in vivo validation of drug targets in mice is still dependent upon funding, decisions on which are pending.  Three of 4 funding agencies are considering our request.  CureSearch passed on funding the dipg preclinical consortium in order to prioritize their Grand Challenge initiatives.  
  
For the manuscript of existing data, a new set of supplemental data by the Grille lab has been created that describes minor but sometimes interesting differences in the way in which tumor cell growth is measured (and is probably worth sharing with the field).   
  
08/01/2013:  Data on pre/post drug treatment (and gene expression changes as a result) is pending runs of the sequencing.  Catie has matured the manuscript, with extensive help and input from Michelle, Ranadip and Jacques (and kind assistance of Lining).  Noah continues to integrate sequencing and drug screening data.  A unifying explanation for how the 2 different drug classes found to be effective might interact is slowly coming to light. Dennis is temporarily taking a leave of absence from the Consortium, with the intention of future active collaborations.  A manuscript submission by the end of the month is the goal (give or take a week), so that the data can be especially freely shared at the September DIPG Trials meeting in Paris, and the October Fall COG meeting.  

08/07/2013:  Catie continues to hone the genomic analysis (the manuscript is to be focused on drug screening results as the genomic changes that correlate them; however, the genomic analysis itself has revealed some rather interesting genes not otherwise on the field's radar).  No other new news at this time. 
  
08/14/2013:  We are moving towards trans-Atlantic clinical trials collaboration that would have complementary, non-overlapping approaches.  Our thanks to Jacques Grille and Darren Hargrave for reaching out in this regard.  Please also see the videos from the Lyla Nsouli Foundation, one of this project's key sponsors.  
  
08/22/2013:  No progress to report this week (on account of Summer holidays).

video
08/28/2013:  Jacques Grill to present results of preclinical consortium at the Sept 2013 NDDS meeting in Paris (organized by Dr. Darren Hargrave).  ps.  This video from the laboratory of Jacques Grille shows how DIPG cells invade a strip of matrigel (mimicking the extracellular matrix seen in a tissue) within a short time frame of less than one day. 
  
  09/05/13:  We were pleasantly surprised with the additional RNA expression studies following treatment with a particular drug in 2 dipg cultures.  There are consistencies with effects of other efficacious drugs... making us feel we might be onto something from multiple lines of evidence.  
  
09/12/2013:  Catie, Nathalene and Marie-Anne continue to work on finalizing figures.  A framework for data sharing and complementary COG, PBTC and SIOP dipg clinical trials is coming together.
  
09/19/2013:  Promising correlation seen between drug effects on gene expression and targets of the original chemical screen. 
  
09/26/2013:  A few normal brain RNA samples found to have contamination with tumor cells to some degree.  Not unexpected, but the process of making figures is now re-started.  
  
10/03/2013:  Awaiting decisions on funding for our 'next steps' proposal for preclinical studies.
 
10/11/2013:  Results presented yesterday at the Children's Oncology Group meeting in Dallas, Texas.  Very well received, and remarkably complementary to current approaches.  A new dipg clinical trial concept, based in part on our consortium's results, was proposed within the group.  Validation experiments in mouse models will be key... and complementarity & transparency with PBTC and SIOP clinical trial groups was again reiterated.  please stay tuned for an exciting funding announcement from our colleagues at a particularly progressive Foundation. 
  
10/18/2013:  Manuscript writing continues, as we try to distill the most interesting details of the consortium project.  The 'next steps' funding to be announced soon, once paperwork is completed with each participating institution. 
  
10/25/2013:  This week begins the big push on manuscript finalization.  A lot of discussions have ensued how best to present the interesting RNAseq data.  Funding announcements on next steps are also still pending.  Details of important pharmacokinetics and pharmacodynamics studies in the next step studies are being worked out. 
  
11/01/2013:  Manuscript writing and figure finalization rapidly underway.  A journal has agreed to review our initial manuscript submission, but a long list of little things (from data finalization to formatting) is still being done. 
  
11/08/2013:  More manuscript writing is being done.  Version control has gone back and forth across the Atlantic.  
  
11/15/2013:  Preparations for the Society of Neuro-Oncology (SNO) meeting presentation are being done for Catie's talk on our results thus far. 
  
11/21-22/2013:  On Wednesday Charles attended a National Brain Tumor Society sponsored special summit at SNO on Availability of Drugs for Pediatric Brain Tumor and Pediatric Cancer Research.  Attendees included industry, parent groups, academic researchers, government regulatory agencies... it was truly a productive meeting, and we hope that great new approaches come to making novel drugs available to children with brain tumors via timely clinical trials.  There remains much work to be done, but the conversation itself was the beginning of this progress.  
  

Catie also presented our dipg preclinical consortium results thus far.  Her talk was well received, and we appreciate the comments afterwards.  Particularly the suggestion to show how dipg cultures responded relative to tumors of other types tested in the same way. 
  
For the 'next steps' funding announcement, now that we have all but one set of paperwork for participating institutions we should be able to make a formal announcement soon.  
  
11/29/2013:  paperwork at all sites now complete so that we can proceed with 'next steps' funding and grant agreement.  manuscript nearly final, but additional validation studies are being done by our European colleagues to ensure differences in culture generation still allow inter-comparison.  
  
12/05/2013:  we still are running additional cell culture validation.
  
12/12/2013:  for one of the drug hits, we now have promising brain pharmacokinetic data.
  
12/19/2013:  for the drug hit, we now have promising in vivo anti-tumor results (likely to be included in manuscript).
  
12/26/2013:  no progress this week (holidays).
  
01/02/2014:  Catie and Michelle are moving forward to finalize the manuscript.  As you might have noticed, manuscript writing has been ongoing in parallel with validation experiments to refine our results since April '13, but this is on par for the kind of study here.  
  

01/09/2014:  Please see the press release on our 'Next Steps' funding, generously provided by the Lyla Nsouli Foundation.  
  
01/16/2014:  Discussions between all labs on harmonization of methods is taking place (thank you Esther, Michelle, Marta and Eric).  Manuscript near-final draft circulated to all authors. New culture model being investigated (thank you, Dennis). 
    
01/24/2014:  Manuscript is submitted and in review at a high impact journal.  Everyone in the consortium deserves acknowledgement for making this possible, especially Catie, Nathalene, Noah, Jacques and Michelle.   
  
01/30/2014:  New DNAs sent from Stanford for sequencing (new samples are really helpful for follow-on informatics studies).  
  
02/06/2014:  ISPNO meeting abstract to be submitted this week. 
  
02/14/2014:  Preliminary discussions started for trans-Atlantic coordinated partnership to determine clinical availability of a promising preclinical agent. 
  
02/20/2014:  New samples/culture to be sent from Steindler lab (Univ. of Florida) to Stanford once material transfer agreement is in place.  Additional experimental samples sent from Monje lab to OHSU for sequencing. 
  
02/27/2014:  We are excited to announce a new, valued collaborator, Dr.  Javad Nazarian, at Children's National Medical Center!  [pictured, from left to right: Sridevi Yadavilli, MD, PhD (postdoctoral fellow); Madhury Kambhampati, MS (technician); Jamila Gittens, MS (technician); Elizabeth Wells, MD (Neurologist); Javad Nazarian (P.I.)] 
  
03/06/2014:  Manuscript will need revisions, including additional experiments, prior to submission to a new journal.  those experiments have been planned amongst the participants.  
  
03/13/2014:  See the NBTS blog post.  
  
03/20/2014:  from the OHSU group (Keller):  we have received more than expected new samples for sequencing (16 new samples from our new consortium collaborator Dr. Javad Nazarian at The George Washington University; 3 new samples from OHSU; and 3 new samples from Stanford). Coordination of projects across the sites is ongoing.
  
03/27/2014:  from the VUMC group (Hulleman):  Initial in vitro HDAC/Wee1 inhibitor studies completed.  Now examining influence of common DIPG mutations on response.  From the JHU group (Raabe): mouse innoculations performed so that preclinical studies can be conducted. 
  
04/03/2014:  from the Navarra group (Alonso): in vivo model characterized recently completed. 
  
04/10/2014:  from the Stanford group (Monje): key CNS pharmacokinetic parameters of selected drugs completed... very promising, looking at it from a given point of view. 
  
04/17/2014:  from the Baylor group (Li):  model expansion and preparations for MRI imaging in progress.   
  
04/25/2014:  preparing for the next batch of tumor DNAs and RNAs for sequencing; synergy studies in biologically distinct cell cultures may be promising for a specific combination. 
  
05/01/2014:  Baylor group to start new preclinical trials soon. 
  
05/08/2014:  adding roughly 25-30 new samples for functional genomics studies to complement preclinical trial analysis
  
05/15/2014:  promising conversations with pharma regarding trial concept with implicated 'hit' from our studies (nothing final); Stanford validation studies of combinations ongoing.
  
05/21/2014:  JHMI group gearing for in vivo studies (multi-week process). 
  
05/28/2014:  One of the combination studies is nearing completion at the Alonso laboratory, with pharmacodynamic studies underway. 
  
06/05/2014:  update from the Baylor team:  a large scale preclinical study of drug A, drug B, or the combination is initiated (will be a multi-week study).  Pharmacodynamic and resistance studies to be incorporated.  
  
06/12/2014:  from the Stanford team:  additional drug combination validations underway using chemical and genetic approaches.  Early results expected in ~2 weeks.   
    
06/19/2014:  Univ. of Navarra update:  dose finding done so that near-final in vivo studies with drug combinations can be conducted. 
  
06/26/2014:  OHSU update:  via consortium sites, an additional 49 new dipg samples for potential DNA exome sequencing and 42 new samples for potential RNA deep sequencing have been collected. We are grateful to the families who have made this possible, many having come from legacy gifts (research autopsies).  
  
07/03/2014:  We very, very much appreciate the $85,000 in supplemental research funding from The Lyla Nsouli Foundation, which makes possible sequencing the above 49 new dipg DNA samples and 42 new dipg RNA samples. These data significantly improve the impact of our ongoing studies, and move us significantly closer to the development of a 2-drug combination for clinical trials of dipg.  
  




  

10 comments:

  1. how can this help newly dx dipg patients?

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  2. hi, Danny. Good question. This study is intended for patients 18-24 months from now, once we have all the results compiled and can design a clinical trial with a high probability of success. So it's not for patients newly diagnosed unless they are alive to enroll in the trial 18 - 24 months from now. I know this is not the answer you want, but we're still aiming to be faster than anything tried before. Charles

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  3. I am very excited about this consoritum. What has the experience been with this type of set up in adults and in pediatric brain tumors with this format?

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  4. hi, Loice. As in the note just sent on the DIPG Yahoo group, the St Jude work by Richard Gilbertson and Kip Guy has informed us a great deal about ependymomas. Keep an eye out, too, for exciting work by Paul Spellman integrating the TCGA Project's genetic data with drug sensitivity... the new frontier in chemo-genomics so to speak. Charles

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  5. I have a nephew who was just diagnosed with DIPG on 7/21/12. When will this study be ready for clinical trials and who do I need to get in touch with?

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    1. Brittany, we will do our best but the timeline of animal studies (needed to justify a clinical trial concept) is at least 6-9 months away as of Jan 2013, and even then clinical trial takes additional months to plan and open. The data will be shared openly with both the Children's Oncology Group (who initiated this research) and the Pediatric Brain Tumor Consortium.

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  6. The ultimate goal of this project was to move the most effective single agent or combination therapy forward to early phase clinical trials in 18-24 months. could you please tell us where are you in the process and when you would expect to be ready for a clinical trial?

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    1. We are about 6-9 months from having mouse (preclinical studies) completed, and even then clinical trial takes additional months to plan and open. The data will be shared openly with both the Children's Oncology Group (who initiated this research) and the Pediatric Brain Tumor Consortium.

      We understand the urgency of your comment, and every lab in the consortium is doing their best to accelerate the research without any corners being cut.

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  7. This new publication came out from one of the consortium institutions. It looks pretty interesting.
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3639279/

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  8. agreed... dipg are notoriously chemotherapy resistant. this could be a prominent factor.

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