Further information

HiPerDART Targets and Objectives

LATEST OBJECTIVES

It is the aim of the HiPerDART project to develop, using a novel printing technology called Supramolecular NanoStamping (SuNS), a higher standard of clinical microarray technology. By both minimizing assay workflow and improving the signal-to-noise ratio of the data, our technology platform promises to dramatically improve the reliability of medical tests to enable the use of this high throughput technology for clinical purposes.

Objectives for the 19th-36th months timeframe can be retrieved from the Annex I document, and they can summarized as follows:

1)   Define a RCA reaction protocol to have suitable templates to be stamped

2)   Identify 10 probes to be spotted in a prototype microarray

3)   Produce a spotted template to be used for array replication and stamping

4)   Produce a “chemistry-adapted” replica surface

5)   Design and produce a hybridization cartridge

6)   Define QC thresholds for Stamping as well as automation of the process

7)   Define a contingency strategy to find newer and better predictors

 

Along with these objectives, we here list a variety of recommendations done from previous reporting and within the 36th month review meeting:

a)   A bigger effort should be dedicated about Dissemination to Scientific Community and General Public (Planning and Actions in D6.4_36 and D6.5_36)

b)   A closer involvement should be dedicated to Advisory Board (Planning in Progress Management paragraph 3)

c)    A gender critic issue has been pointed out during the 36th month review, about Management Board (Actions in Progress Management paragraph 3)

d)   Upon reporting, a clear explanation of the different contributes within WPs has to be given;

e)   More information should be given on how the Consortium will exploit the technological outcome (Actions in Project Management paragraph)

2. Work progress and achievements during the period

The aim of this chapter is to describe in details the progress achieved by each single work group and to illustrate the achievements and the status of the activities with major focus on the 19-36th month period.

The present document presents in details the status of the project including the deliverables and milestones tables and their accomplishment degree respect to scheduled plan. A review of the experimental results is given, along with the different contributes from each partner. A summary of the progresses will be given towards objectives for each task. Detailed experimental procedures and data analysis have been uploaded in the HyPerDART website as requested by WP6 deliverables. The report is updated up to December 30th 2011.

Each single deliverable has been uploaded in the electronic submission website, and, where applicable, a PDF attachment has been uploaded separately to both give experimental details and better explain any deviation from the original Annex structure.

Following the presented objectives in paragraph 1, we here report a concise overview of the progress of the work:

RCA reaction in solution has been optimized and the ideal protocol to produce RCA polymers starting from dNTP and a circular sequence has been set up; an extensive study on the RCA reaction on glass has been done in parallel; the presented results showed that RCA reaction on glass does occur, however the minimum intensity threshold for SuNS applications has not been reached. For this reason, the preparation of RCA templates by spotting polymerized sequences (in solution), has been followed in the last 3 months of the reporting period. 10 probes has been identified as possible predictors; these sequences have been spotted in a prototype microarray, as decided during the first 18 months project review. The spotted templates are being used for array replication on glass and for direct hybridization of real samples from colon cancer. Stamping experiments are on on-going, and involve the use of both model replica surfaces and EPFL polymer brush layers.

After a careful revision of the predictors, a newer strategy has been identified; Next Generation Sequencing will be used to find a more efficient predicting set of probes. This holds as contingency plan for both WP5 and WP2 activities.

Polymer brush layers have been designed and synthesized after an initial delay due to technical problems; a bigger effort has been dedicated to functionalize the obtained and characterized layers for bio-molecule immobilization. Aldehyde functionality has been introduced, and the capability of these layers to link oligonucleotide sequences has been successfully verified. The possibility to grow active polymer brushes on PDMS sub-layers is being currently evaluated.

A first generation cartridge has been realized and tested during the first reporting period; a Second Generation Cartridge has been designed after that, and the major drawings are presented in this report.

Model templates have been used to define the stamping QC thresholds; a detailed study on stamping has been carried out involving different stamping under-layers, fluorophores and mechanical stamping parameters. As a significant results, a set of QC thresholds has been found. On the other hand an intense activity on stamping automation has been carried out. New tools for automated loading and unloading of the slides have been designed and realized.

A bigger effort about dissemination activities has been done by the whole consortium. The scientific production in terms of papers, oral communications and participation at relevant conferences has been presented within this report. Following the useful suggestion made by the European Officer during the 36th month review meeting, a new website has been created to increase communication toward general public and clinical community.

We recall that the HiPerDART project has been structured in 6 work packages whose responsible are:

ID

Responsible

WP1

Project Management

MS

WP2 Template Production

KTH

WP3 Replica Surface Production

EPFL

WP4 SuNS Technology Development

MS

WP5 Validation and Development of Prognostic Predictor

ICO

WP6 Dissemination

MS

WP Names and Responsibilities

Deliverables Table

Del. no.

Deliverable name

WP no.

Lead  beneficiary

 

Nature

Dissemination
level

 

Delivery date from Annex I (proj month)

Actual / Forecast delivery date

Dd/mm/yyyy

Status

No submitted/

Submitted

Comments

1.1

Consortium agreement

1

1

R

PP

month 3

04/12/2008

Submitted

File D1.1 Consortium Agreement

1.2

Quality Assurance & Risk analysis

1

1

R

PP

month 6

30/06/2009

Submitted

File D1.2 Quality Assurance

1.3

Progress reports

1

1

R

PP

month 12, 18, 24, 36

30/12/2011

Submitted

File D 1.3

Progress Reports.

Additional file containing progress report for month 24, File D1.3_36 Progress Reports

1.4

Progress Meetings

1

1

R

PP

month 18, 36

06/07/2010,

16/12/2011

Submitted

File D1.4 ProgressMeeting July2010 LausanneFile D1.4_36 Progress Meeting Dec 2011 Barcellona

1.5

Reports to EC

1

1

R

PP

month 18, 36

 

Molecular Stamping is issuing the present report as 36th month review to EC

1.6

Audit Certificates

1

1

R

PP

month 18, 36

1.7

Final report to EC

1

1

R

PP

month 48

This deliverable did not start yet

2.1

Production of single sequence DNA Array

2

2

P

PP

month 5

08/05/2009

Submitted

Completed. File D2.1 ProductionDNA

2.2

Production of single sequence LHD array via RCA

2

2

P

PP

month 9

10/10/2009

Submitted

Completed. File D2.2 ProductionLHD

2.3

Production of confined single sequence DNA array

2

2

P

PP

month 10

30/09/2010

Submitted

3 months delay vs. forecast; File: D2.3 Production of confined single sequence DNA array.

The RCA reaction on the array did not work enough for the SuNS intensity signal requirements; experimental data have been presented and contingency plan explained in File D2.3_36

2.4

Production of  confined DNA array

2

2

P

PP

month 15

Fcst 30/09/2012

Submitted

File: D2.4 Confined DNA array

This deliverable refers to Array Replication; the due date has been delayed due to first available spotted microarrays in Dec 2011. File D.24_36

2.5

Production of confined LHD array

2

2

P

PP

month 18

Fcst 30/06/2012

Submitted

File D2.5Production LHD.

This deliverable refers to the preparation of the spotted template instead of the RCA reaction on the array. The final optimization of the spotting should be completed by June 2012. File D2.5_36

2.6

Optimization of LHD array

2

2

P

PP

month 30

Fcst 30/09/2012

Submitted

This deliverable refers to the optimization of the spotted prototype. The final spotted array will include the probes from NGS method; this should be done by Sept 2012. File D2.6_36

3.1

Replica surfaces modified with active ester functionalized PPEGMA coatings of different thicknesses.

3

3

R

PP

month 6

10/07/2009

Submitted

Completed. A basic characterization of non-functionalized polymer brushes has been achieved. File: D3.1 ReplicaSurface with PPEGMA

3.2

Replica surfaces modified with active ester functionalized PPEGMA coatings that are applied on a microstructured elastomeric PDMS sublayer

3

3

R

PP

month 18

Fcst 30/06/2012

Submitted

File: D3.2 Replica surface with PPEGMA.

Aldehyde functionalized Polymer brushed have been found as suitable layers for immobilization of bio-molecules; this deliverable is delayed till these aldehyde layers have been fully characterized. In parallel preliminary attempts to functionalize oxidized PDMS layers with polymer brushes just started. D3.2_36

3.3

Replica surfaces modified with active ester functionalized PPEGMA coatings that are applied on a polyacrylamide hydrogel sublayer

3

3

R

PP

month 24

Fcst 30/09/2012

Submitted

Aldehyde functionalized Polymer brushed have been found as suitable layers for immobilization of bio-molecules; this deliverable is delayed till these aldehyde layers have been fully characterized. D3.3_36

3.4

Replica surfaces modified with hyperbranched PPEGMA coatings of different thicknesses.

3

3

R

PP

month 30

Fcst 30/09/2012

Submitted

This deliverable has not been addressed yet, The outcome of the studies of aldehyde layers will determine the necessity of studying the hyperbranched polymer brush system. File D3.4_36

3.5

Replica surfaces modified with PPEGMA coatings presenting different surface concentrations of vinyl sulfone, maleimide, azide or acetylene functionalities

3

3

R

PP

month 36

Anticipated

30/06/2011

Submitted

Anticipated to month 30th.

Aldehyde functionalized layers have been prepared and its basic characterization is almost completed. File D3.5_36

3.6

Report on the specifications of the hybridization set-up and concept of cartridge and set-up

3

3

R

PP

month 6

30/06/2009

Submitted

File: D3.6 specifications of the hybridization set-up and concept of cartridge and set-up

3.7

First generation of cartridge and test set-up

3

3

R

PP

month 24

30/01/2011

Submitted

File D.3.7_36. First generation of cartridge and test set-up

3.8

Replica surface modified with optimized hydrogel coating

3

3

R

PP

month 48

30/12/2012

No Submitted

This deliverable did not start yet

3.9

second generation of cartridge

3

3

R

PP

Month 48

30/12/2012

Submitted

The second generation cartridge has designed and prototyped. The rest of the project will be dedicated to its complete development. File D3.9_36

4.1

Engineering design of instrument from Advanced Automation

4

1

P

PP

month 3

12/02/2009

Submitted

Drawings available in CAD format

File D4.1Eng_design_Advance_Automation

4.2

Stamping instrument passes site acceptance test

4

1

R

PP

month  8

26/11/2009

Submitted

Conditional released in July 09; full release Nov 26th 2009

File: D4.2 AA_SAT

4.3

DNA transfer measurements as function of process variables

4

1

R

PP

month  18

30/06/2010

Submitted

File: D4.3 DNA Transfer Measurement

4.4

QC definitions; first product passes QC

4

1

R

PP

Month 24

30/09/2012

Submitted

File D4.4_36 The basic QC parameters and thresholds have been found; the final validation of these parameters is still on-going due to the recent availability of HiPerDART templates to be stamped.

4.5

Design for integrated and automated industrial production equipment

4

1

R

PP

Month 34

30/05/2012

Submitted

File D4.5_36

5.1

Databases with information to build the predictors from existing data

5

5

O

PP

month 7

06/08/2009

A total of 1482 expression arrays from 9 studies (7 public and 2 proprietary) has been collected. Dataset clinical information stored in WP5 documents site at http://hiperdart.fbk.eu.

File D5.1 Database

5.2

Hybridization and analysis of additional data for CNV and expression in normal mucosa

5

5

R

PP

month 9

Fcst 30/10/2010

Submitted

Delay due to the high selection criteria to get a homogenized sample of stage II patients without neo-adjuvant treatment, only with stable in microsatellites cases, high quality of the sample frozen in a tumor bank and verified clinical follow up for almost three years. Additionally it has been slow to recruiting 50 healthy patients who wish to participate in the study and for which informed consent is mandatoryFile: D5.2 Hybridization CNV.

Additional file about completition D5.2_36; NGS method as new predictor tool.

5.3

Data mining results with the genetic profiles identified to have best predicting ability for each

5

5

R

PP

month 11

08/12/2009

Submitted

Completed.

File: D5.3 DataMiningResults

5.4

The set of probes needed to be stamped in the SuNS microarray for further validation

5

5

P

PP

month 13

Fcst

30/11/2010

Submitted

Additional effort needed.

File: D5.4 SetOfProbes. Additional file about completition D5.4_36

5.5

Preparation of RNA samples from the MAQC panel

5

5

R

PP

month 15

Fcst  30/06/2011

submitted

ICO is working on the accurate clinical selection of samples for the validation on the MAQC panel. When the selection is fully curate and validated with the ICO bio bank stock will begin with the RNA extraction.

File D5.5 Preparation of RNA. Additonal file about completition D5.5_36

5.6

Hybridization of  SuNS microarrays

5

5

R

PP

month 20

30/06/2012

No Submitted

Depends on SuNS availability

5.7

The measurements QC and normalization

5

5

R

PP

month 20

30/06/2012

No Submitted

Depends on SuNS availability

5.8

The analysis of concordance and assessment of validity and precision

5

5

R

PP

month 24

30/06/2012

No Submitted

Depends on SuNS availability

5.9

Selection of samples, dissection for tumor cell enrichment or normal mucosa and extraction of RNA and genomic DNA for the different assays proposed

month 24

30/06/2012

Submitted

Details about the degree of completition and work done in D5.9_36

5.10

Hybridization of  SuNS microarray for 500 samples

5

5

R

PP

month40

No Submitted

Not Started yet

5.11

The measurements QC and normalization

5

5

R

PP

month44

No Submitted

Not Started yet

5.12

Statistical analysis for the validation of the prognostic profile

5

5

R

PP

month48

No Submitted

Not Started yet

6.1

Scientific Launch  Meeting

6

1

O

PP

month  3

18/02/2009

Submitted

File: D6.1 Minute_KOM Hiperdart

6.2

Online  Data management tool /website

6

1

O

PP

month  3

30/03/2009

Submitted

File: D6.2 OnlineDataWebsite

6.3

Project updates

6

1

R

PP

month 3

30/03/2009

Submitted

Project update has been given periodically to all involved parties by emails, by conference call end by posting information’s  on the web site.

File: D6.3 Project_updates

6.4

Programme for Dissemination of information

6

1

R

PU

month 6

30/06/2009

Submitted

File: D6.4 Dissemination program

Additional Efforts described in D6.4_36

6.5

Symposia and Workshops

6

1

R

PU

month 24

30/12/2012

Submited

Addtional efferts have been done to compensate on Dissemination within General Pubblic, Scientific Community, Sclinical Community. File D6.5_36

6.6

International Conference

6

1

R

PU

month 48

30/12/2012

No Submitted

This deliverable did not start yet

Milestones Table


List and schedule of milestones

 

Milestone no. Milestone name WPs no’s. Lead beneficiary no. Delivery date from Annex I Comments
1 Template WP1, WP2 2 Month 12 Major problems have occurred during the development of RCA on the Array; experimental data supporting this observation have been given in file D2.3. A contingency plan has been decided, ie, to prepare the polymeric RCA probes in solution, and to spot the obtained polymer on glass; details about this plan have been presented in file D2.5. This contingency plan lead the Consortium to get to a suitable template in November 2011.
2 Hybridisation WP1, WP2, WP3 3 Month 24 The replica surface has been found (see details in D3.5); the basic characterization has been done; stamping experiments are on-going, and they strongly depend on achievements arising from Array Replication Development (D2.4). The hyb Cartridge has been defined and needs to be tested (D3.9)
3 Replica WP1, WP2, WP3, WP4 1 Month 24 Array replication is on-going; the first stamping tries have just started.
4 Standard Test WP1, WP5 5 Month 36 HiPerDART microarray evaluation started with standard set and by using the spotted template.
5 Diagnostic Test WP1, WP5, WP6 1 Month 48 Real colorectal cancer samples are being used to hybridize the spotted templates.

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