DELIBERAZIONE N. 602/2015 ADOTTATA IN DATA 16/04/2015 · 2015-04-20 · The new HTS based method...

Responsabile del procedimento dr.ssa Mariagiulia Vitalini U.S.C. Affari Generali MGV

FIRMATA DIGITALMENTE dal Direttore generale / Direttore sanitario / Direttore amministrativo

OGGETTO: Autorizzazione allo svolgimento presso l’USC Ematologia del progetto diricerca “High Throughput Sequencing efficacy in clonality and MRD evaluationin ALL patients lacking molecular probes by ASOQPCR” approvato efinanziato dall’Associazione italiana per la ricerca sul cancro (AIRC).

IL DIRETTORE GENERALE

Assistito dal Direttore sanitario e dal Direttore amministrativo, che svolge le funzioni diverbalizzante;

Premesso che:

in data 18 marzo 2014 è stata inoltrata all’Associazione italiana per la ricerca sul cancro(AIRC), nell’ambito della Call for Proposal 2014, la richiesta di finanziamento del progetto“High Throughput Sequencing efficacy in clonality and MRD evaluation in ALL patientslacking molecular probes by ASOQPCR” proposto dall’USC Ematologia - Laboratorio didiagnostica ematologica “Paolo Belli”;

con nota del 18 novembre 2014, AIRC ha reso noto che il Consiglio direttivo, avendovalutato positivamente il progetto, lo ha approvato chiedendo nel contempo larimodulazione delle voci di budget, atteso che a fronte di una spesa prevista di € 245.718,00è stata messa a disposizione dell’azienda la somma complessiva vincolata di € 240.000,00per il triennio 2015 - 2017;

in data 12 gennaio 2015 è stato quindi dato riscontro alla richiesta di AIRC, trasmettendo lascheda di rimodulazione del budget triennale;

Premesso, altresì, che con scritto in data 10 marzo 2015, prot. n. 9973, il direttoredell’USC Ematologia ha chiesto l’autorizzazione a condurre presso il Laboratorio didiagnostica ematologica “Paolo Belli” il citato progetto di ricerca e ha trasmesso,contestualmente, la relativa documentazione, dalla quale si rilevano le caratteristiche delprogetto di ricerca, documentazione alla quale si rinvia per gli eventuali approfondimenti;

Sottolineato l’interesse di questa azienda alla conduzione del citato progetto, atteso cherientra tra le proprie finalità lo svolgimento di attività di ricerca;

Accertato che il progetto rientra tra le collaborazioni scientifiche che prevedono losvolgimento di attività di studio in ambito laboratoristico, con l’utilizzo di campioniprovenienti da pazienti che già hanno fornito il consenso alla raccolta, stoccaggio e impiego dicampioni biologici per finalità di ricerca, nel rispetto delle prescrizioni del Comitato dibioetica;

DELIBERAZIONE N. 602/2015 ADOTTATA IN DATA 16/04/2015

Responsabile del procedimento dr.ssa Mariagiulia Vitalini U.S.C. Affari Generali MGV

FIRMATA DIGITALMENTE dal Direttore generale / Direttore sanitario / Direttore amministrativo

Accertato, altresì, che la tipologia della collaborazione non richiede l’acquisizione di unapreventiva autorizzazione da parte del Comitato di bioetica ma la sola notifica allo stessoComitato ai fini della presa d’atto del progetto di ricerca;

Ritenuto, pertanto, di poter accogliere la richiesta del direttore dell’USC Ematologia;

Ritenuto, altresì, al fine di semplificare le fasi di riscossione e di utilizzo delfinanziamento AIRC vincolato alla realizzazione del progetto di ricerca “High ThroughputSequencing efficacy in clonality and MRD evaluation in ALL patients lacking molecularprobes by ASOQPCR”, di autorizzare i competenti uffici a introitare le somme che AIRCassegnerà annualmente al progetto di ricerca, destinandole al fondo di struttura dell’USCEmatologia al netto della quota destinata alla copertura dei costi indiretti e dei costi generali,nella misura prevista dalla scheda di “Rimodulazione budget”;

Ritenuto, inoltre, per le medesime finalità, di porre in capo al direttore dell’USCEmatologia la responsabilità di utilizzare la quota di finanziamento che alimenterà il fondo distruttura nel rispetto del budget rimodulato di progetto e delle eventuali istruzioni di AIRC perla gestione amministrativa del grant assegnato nonché del regolamento per la gestione dei fondidi struttura, approvato con deliberazione n. 453 del 13 aprile 2010;

DELIBERA

1. di autorizzare l’USC Ematologia a condurre il progetto di ricerca “High ThroughputSequencing efficacy in clonality and MRD evaluation in ALL patients lacking molecularprobes by ASOQPCR” approvato e finanziato dall’Associazione Italiana per la Ricerca sulCancro (AIRC) (allegato A);

2. di prendere atto della scheda “Rimodulazione budget”, dalla quale si rileva che all’aziendaè destinato un finanziamento complessivo triennale di € 240.000,00, suddiviso in annualitàdi ammontare pari a € 80.000,00 (allegato B);

3. di autorizzare i competenti uffici ad introitare annualmente il finanziamento AIRCdestinandolo al fondo di struttura dell’USC Ematologia, al netto della quota relativa allacopertura dei costi indiretti e dei costi generali, nella misura indicata dalla scheda di“Rimodulazione budget”;

4. di porre in capo al direttore dell’USC Ematologia la responsabilità di utilizzare tale sommanel pieno rispetto del budget rimodulato di progetto e delle eventuali istruzioni di AIRC perla gestione amministrativa del grant assegnato nonché del regolamento sulla gestione deifondi di struttura, approvato con deliberazione n. 453 del 13 aprile 2010.

IL DIRETTORE GENERALEdott. Carlo Nicora

IL DIRETTORE SANITARIO IL DIRETTORE AMMINISTRATIVOdott.ssa Laura Chiappa dott. Peter Assembergs

AIRC

Associazione Italiana per la Ricerca sul Cancro

Investigator Grant - IG 2014

YEAR 2014

Current address: Via San Vito, 7 - 20123 Milan, Italy

Legal address: Via Corridoni, 7 - 20122 Milan, Italy

Tel: +39-02-7797-374/350/222

Fax: +39-02-7797259

E-mail: [email protected]

Table of Contents

TITLE PAGE 3

ABSTRACT 4

PROPOSAL MAIN BODY 5

PERSONNEL INVOLVED IN THE RESEARCH 14

DESCRIPTION OF THE WORK FOR EVERY UNIT OF PERSONNEL 15

BUDGET FORM AND JUSTIFICATIONS 16

BIOGRAPHICAL SKETCH 18

NARRATIVE BIOSKETCH 19

PI TRACK RECORD SUMMARY 20

PUBLICATIONS OF THE PI 21

BIO-ETHICAL REQUIREMENTS 28

Addendum A - PERSONNEL INVOLVED IN THE RESEARCH - CURRICULUM VITAE 29

Addendum B - BUDGET FORM AND JUSTIFICATIONS 31

Addendum C - PUBLICATIONS OF THE PI 32

Addendum D - BIO-ETHICAL REQUIREMENTS 33

TITLE PAGE Principal Investigator

SurnameRambaldi

NameAlessandro

PositionHead

Proposal Title

High Throughput Sequencing efficacy in clonality and MRD evaluation in ALL patients lacking molecular probes by ASO-QPCR

Type of grant IG 2014

Area Cancer Genetics

Keywords ALL; Minimal Residual Disease (MRD); Next generation sequencing

Budget 2014 (euro): € 81.906,00 Estimated budget 2014 - 2016 (euro): € 245.718,00

Hosting Institution Department/Laboratory

Azienda Ospedaliera Papa Giovanni XXIII - "Paolo Belli" Laboratory

AddressPiazza OMS - Organizzazione Mondiale della Sanità, 1

Zipcode and City24127 Bergamo (Bergamo)

Phone0352674649 Fax E-mail

[email protected]

Legal Representative

Nicora Carlo

AddressPiazza OMS - Organizzazione Mondiale della Sanità, 1

Zipcode and City24127 Bergamo (BG)

Phone Fax [email protected]

Proponent's signature Legal Representative's signature Date_____________________ _____________________ 18 mar 2014Rambaldi Alessandro Nicora Carlo

TITLE PAGERambaldi Alessandro [PI]

Codice Riferimento: 16105

ABSTRACT BackgroundResidual leukemia levels (Minimal Residual Disease, MRD) has been demonstrated as the most important predictor of outcomeof Acute Lymphoblastic Leukemia (ALL) patients (1, 2). From year 2000 two multicenter clinical trials (NILG-09/00,ClinicalTrials.gov Id: NCT00358072 and NILG-10/07 ClinicalTrials.gov Id: NCT00795756) have been conducted to testprospectively the predictive value of MRD in adult ALL patients (3). Patients with undetectable MRD (MRDneg) at the end ofinduction/consolidation received maintenance chemotherapy while patients with persistence of disease (MRDpos) receivedallogeneic transplantation or high-dose chemotherapy supported by autologous transplant. The MRDneg cohort had a DFS of72% while MRDpos cohort had a DFS of 14% at 5 years (3). A proportion of patients (MRDunk) could not benefit of MRDbased treatment allocation due to the lack of a molecular probe with a suitable sensitivity, as reported for adult (4) and pediatric(5) ALL patients. In our studies MRD evaluation was performed with allele-specific oligonucleotide quantitative PCR (ASO-QPCR) which is considered the gold standard method (6). This approach requires the development of reagents and assayconditions for each patient and is laborious and time consuming. Recently, it has been described a new approach based on HighThroughput Sequencing (HTS) to identify clonal rearrangements and to monitor MRD in ALL and lymphoid malignancies (7,8). This approach does not need the development of patient specific reagents, applies the same strategy at diagnosis andmonitoring and can release molecular information suitable for clinical decision in as few as 7 days.

HypothesisThe new HTS based method could be useful to identify clonality and to monitor MRD in patients in which gold standard ASO-PCR failed.

AimsAim of this study is to apply the HTS technology to patients enrolled into our clinical trials that could not benefit of MRDcategorization due to the lack of a molecular marker suitable for MRD evaluation. The new categorization as MRDpos orMRDneg derived from this study will be correlated to patients’ outcome.

Experimental DesignHTS technology validation will be performed on ten diagnostic and ten post induction/consolidation samples completely andsuccessfully characterized for clonality and MRD evaluation with conventional ASO-QPCR. Subsequently, HTS technologywill be applied to samples of patients who failed MRD characterization (MRDunk) with conventional method. MRD re-categorization in MRDpos or MRDneg will then be correlated to patients’ outcome. Finally the HTS technology will beprospectively applied to the MRDunk cohort of patients identified in the new, national treatment protocol (ClinicalTrials.gov Id:NCT02067143).

Expected ResultsWe expect that HTS technique applied to the validation set will obtain results substantially concordant with those previouslyobtained with a possible increase in identified clonal marker at diagnosis. MRD evaluation should also be concordant with someexceptions: low positive reclassified as background and negative reclassifies as low positive due to the HTS higher specificityand sensitivity. We also expect to be able to find clonal rearrangements and to monitor MRD in the MRDunk cohort of patientsof the previous as well as new prospective clinical trials.

ABSTRACTRambaldi Alessandro [PI]

Codice Riferimento: 16105 Page 4 of 39

Background

Acute Lymphoblastic Leukemia (ALL) is a rare lymphoid malignancy characterized by an

aggressive clinical course in adults patients. Treatment of ALL consists of remission induction,

consolidation and maintenance chemotherapy with or without allogeneic stem cell transplantation.

Complete remission (CR) can now be achieved in the majority of adults with ALL but only 40-50%

is cured, the proportion varying according to well-described clinical and biological risk factors. The

main adverse risk factors are: older age (>35-55 years), hyper leukocytosis (>25-100x109/L), time

to complete remission (> 4 weeks), and adverse cytogenetic abnormalities such as t(9;22) and

t(4;11). It has been demonstrated that the measurement of residual leukemia levels (Minimal

residual disease, MRD) represent the most important predictor of outcome as the persistence of

MRD is the strongest adverse prognostic factor in this disease (1, 2).

Current methodologies for MRD monitoring include flow cytometry detection of aberrant

immunophenotype and allele-specific oligonucleotides quantitative PCR (ASO-QPCR)

amplification of immunoglobulin (Ig) and T-cell Receptor (TCR) genes. Flow cytometry can detect

1 leukemic cell among 10.000 normal cells (i.e. 1x10-4

detection limit) and requires a high level of

operator expertise to interpret results. ASO-QPCR approach is usually more sensitive and can reach

reproducible detection limit of up to 1x10-5

(i.e. 1 leukemic cell among 10.000 normal cells) (9).

This technique is based on the amplification of the most commonly used TCR and Ig

rearrangements followed by Sanger sequencing. The obtained sequences are used to design allele

specific oligonucleotides (ASO) on junctional regions that are unique for each lymphocyte

rearrangement. These oligonucleotides are then used for MRD evaluation by quantitative PCR in

combination with primers and probes recognizing constant regions of the identified rearrangement.

ASO-QPCR conditions have to be optimized for each found rearrangement in each patient to find at

least two molecular markers with sufficient (10-4

) sensitivity. This procedure requires time, efforts

and specialized personnel and its applicability is restricted to patients with Ig/TCR gene

rearrangements with junctional regions suited to reach sufficient sensitivity. Another important

limitation of the above mentioned techniques is the limited or absent capability to identify the

presence of oligoclonality at diagnosis or to monitor the clonal evolution of the leukemic clone that

can generate potential false negative MRD evaluation during the follow-up of the patients (10).

Recently, it has been described a new approach to identify clonal rearrangements and to monitor

minimal residual disease in Acute lymphoblastic leukemia and lymphoid malignancies (7, 8). This

method is based on the amplification of all the known genes of Ig and TCR loci and the sequencing

of the amplification products on a High Throughput Sequencing (HTS) platform. With this

technique it was possible to identify 1-6 clonal markers per patient and monitor the MRD with a

PROPOSAL MAIN BODYRambaldi Alessandro [PI]


sensitivity of 1x10-6

. Furthermore, a good correlation was seen between the standard clonal

rearrangement identification (6) and the new technique. The same correlation was seen between the

MRD results obtained with the patient specific approach (6) and the new method; discrepancies was

mostly due to the higher sensitivity of the HTS based technique and on its capacity to identify

clonal evolution of leukemic cell (8). The advantage of this approach is that it does not need the

development of patient specific reagent but it applies the same strategy to all patients in all the

disease phases, i.e. diagnosis and monitoring and can release molecular information suitable for

clinical decision in as few as 7 days. Furthermore, due to the possibility to obtain many individual

sequences even in follow-up samples, this methodology can well discriminate between leukemia

rearrangements and similar normal rearrangements which can generate background amplification in

ASO-QPCR and cause difficult results interpretation.

However this promising technology has been applied to selected cases of B precursor ALL and B

lymphoid malignancies and the authors suggest the usefulness to apply this method in prospective

studies to evaluate predictive value of HTS in MRD detection (8). Furthermore, other similar HTS

based approaches are under development within international cooperative groups

(EuroClonality/EuroMRD). This new technology, upon validation by scientific community, could

easily represent the future for minimal residual disease detection for patients affected by acute

lymphoblastic leukemia. In addition, it could represent a valid alternative from now for patients in

which no appropriate probe can be generated with standard procedure (6).

In our hospital starting from 2000 year a multicenter treatment study for ALL (NILG trial 09/00,

ClinicalTrials.gov Id: NCT00358072) was initiated to test prospectively the predictive value of

minimal residual disease (MRD) in adult ALL patients (n=280) (3). Our laboratory centralized

samples for all the participating institutions and developed a molecular probe based on clone-

specific T cell Receptor (TCR) or Immunoglobulin (Ig) rearrangements. With this approach we

were able to categorize patients in two distinct groups after the consolidation therapy: one in which

the minimal residual disease was undetectable (MRDneg) and one with detectable residual disease

(MRDpos); the first group of patient received only chemotherapy whether the second group was

switched to an allogeneic stem cell transplantation, when possible or an high dose chemotherapy

course supported by an autologous stem cell transplantation. The MRDneg group had a DFS of

72% weather the MRDpos had a DFS of 14% at 5 years (3). A proportion of patients (30 out of 142

patients concluding the consolidation phase in our cohort) could not benefit of MRD categorization

for subsequent treatment allocation. This was mainly due to the lack of a molecular probe with a

suitable sensitivity. This condition is common to adult ALL patients (3) (4) as well as pediatric

ALL patients (5). Due to the 09/2000 as well as the 10/07 (ClinicalTrials.gov Id: NCT00795756)



clinical trial a high number of well characterized samples are present in our Institution suitable for

the verification of the performance of the HTS based technologies in identifying clonal

rearrangements and monitor MRD especially in patients with no molecular probes identified by

conventional approach (6). All the patients signed an informed consent for samples collection,

clonal rearrangements identification and for the MRD evaluation. Clinical and biological features

are also registered into databases.

Furthermore a new, national MRD based study (ClinicalTrials.gov Id: NCT02067143) will be

started at our institution in which our laboratory will be one of the three reference laboratory for

rearrangement probe identification and MRD evaluation. In this context it will be possible to apply

prospectively the HTS technology patients in which conventional methodology failed to identify

molecular probes suitable for MRD evaluation.

Experimental Design

Since year 2000 all the patients affected by acute leukemia and suitable for enrolment into a clinical

trial were included and treated accordingly. In 09/2000 clinical trial (ClinicalTrials.gov Id:

NCT00358072) treatment options for ALL were decided based on MRD analysis performed on

bone marrow samples collected during the induction and consolidation phase. Patients with

persistence of MRD (MRDpos) underwent allogeneic transplantation, when possible, or high dose

chemotherapy supported by autologous stem cells transplant. Patients with no detectable MRD

(MRDneg) were consolidated with two year chemotherapy (3). Two hundred and thirty six patients

out of 280 enrolled patients reached the hematological remission and 146 completed the induction

and consolidation therapy. For 112 of the latter group the MRD evaluation was possible with MRD

positivity found in 54 patients and MRD negativity in 58 patients who were treated accordingly.

Thirty patients had no suitable molecular probe (3).

To validate the HTS method 10 diagnostic samples will be used. These samples will be selected

between the MRDpos patients (n=5) and MRDneg patients (n=5) in which at least two sensitive

probes (10-4

) were obtained. The number and the type of clonal TCR and Ig rearrangements

obtained by HTS will be compared to the rearrangements found with conventional techniques (6).

Subsequently, the MRD time points collected at the end of consolidation and used to categorize

these patients as MRDneg or MRDpos for treatment allocation will also be evaluated with HTS

techniques to measure residual disease and to compare qualitative (pos/neg) and quantitative (MRD

levels) results with those obtained with conventional ASO-QPCR. These follow-up samples will be

selected with different MRD levels (from neg to pos) including a case in which, after the MRDneg



classification, a relapse was registered due to a subsequent, documented clonal evolution. We

expect to substantially confirm results obtained with conventional technique with possibly an

increased number of molecular markers found. We also expect to confirm MRD positive results on

follow-up samples with some exceptions in low ASO-QPCR positivity where some of them could

be re-defined as background amplification from normal leucocytes. Conversely, MRD negative

sample re-evaluation could lead to much more different results. In fact the new HTS method,

claimed to be more sensitive (down to 1x10-6

) than traditional (1x10-5

), could identify patients with

persistence of residual disease that could give rise of some relapse occurred in the MRD negative

cohort.

Upon technology validation on well characterized samples, the cohort of MRD unknown

patients (n=30) will be subjected to HTS study. Diagnostic samples will be used to identify clonal

rearrangements and stored follow-up samples will be used for MRD evaluation. Additional samples

from MRDunk patients of the subsequent 10/07 trial could be used to enlarge the study cohort, if

needed. We expect to be able to identify one or more clonal rearrangements in these patients and to

re-classify patients as MRDpos or MRDneg; the outcome of these patients will be correlated to this

new classification. The percentage of success of the technique in MRDunk patients will also

contribute to clarify if the failure in identifying a suitable clonal rearrangement relies on the

limitations of the conventional method firstly applied (6) or if it identifies a group of genetically

immature ALL with low rearranged receptors. This latter hypothesis seems not to be supported by

immunophenotypic features of this cohort, but nevertheless it need to be formally demonstrated.

Finally, we will prospectively use this new HTS based technology on patients enrolled into the new,

national MRD based study (ClinicalTrials.gov Id: NCT02067143) for ALL treatment. In this

clinical trial all the patients will be studied with conventional techniques and only patients with no

identification of molecular probe suitable for MRD analysis will be analyzed with HTS based

technologies. Subsequently, one decisional time point follow-up sample per patient (end of

consolidation) will be studied with the new technique to categorize patients into the MRDneg or

MRDpos cohort and to be later correlated to clinical outcome. This latter point could demonstrate

the feasibility of such approach in a prospective way where rearrangement identification and MRD

results should be available as soon as possible to drive clinical decisions.

Tasks:

Task 1-Analysis with HTS technique of the residual diagnostic material of a small cohort of 10

patients enrolled into the ALL 09-2000 and categorized as MRDpos or MRDneg.



1.1 Sequencing of the diagnostic material of 5 MRDpos and 5 MRDneg patients on the HTS

platform

1.2 Analysis of the output sequences to identify Ig/TCR gene rearrangements

1.3 Comparison between Ig/TCR gene rearrangements identified by the new HTS platform and by

the conventional EuroMRD approach.

1.4 HTS sequencing of the end of consolidation samples of the same10 patients and comparison of

MRD results with those obtained with conventional AOS-QPCR.

Task 2- HTS analysis of the available diagnostic material of the MRD unknown cohort of patients

enrolled into the ALL 09-2000 study and treated following the clinical risk score because of a lack

of a molecular marker.

2.1 Sequencing of the diagnostic material of MRDunk cohort of patients on the HTS platform

2.2 Analysis of the output sequences to identify Ig/TCR gene rearrangements

2.3 Outcome analysis of MRDunk cohort newly re-classified as MRDpos or MRDneg.

Task 3- Prospective application of the HTS technique in parallel with conventional method on

newly diagnosed ALL patients which failed clonal rearrangement identification with conventional

approach.

3.1 Prospective Ig/TCR gene rearrangements identification with new HTS technique.

3.3 Analysis of MRD on samples collected at the end of consolidation therapy with the new HTS

method.

3.4 Evaluation of feasibility and applicability of this analysis in a prospective clinical trial in which

treatment are decided based on MRD results.

Feasibility

Material availability

During the 09-2000 and 10/07 clinical studies diagnostic and follow-up samples were collected,

studied and cryopreserved upon informed consent sign. This material is available for the proposed

study. All the biological features and significant clinical information were also collected into a data

file, which is accessible for the study. All newly ALL diagnosed patients will sign an informed

consent before enrollment into the new, prospective national trial (ClinicalTrials.gov Id:

NCT02067143).



Experience of the lab

Our laboratory is experienced in cytomorphologic and immunophenotypic diagnosis of hematologic

diseases from 1987. Molecular diagnosis was introduced since 1994 for qualitative PCR and since

2004 for quantitative PCR. It is composed by a Senior PhD, two technicians, three senior fellows

and five younger fellows. Our laboratory was involved in the standardization of the qualitative (11)

and quantitative (12) analysis of the most common translocations in Acute Leukemia (LA) within

the BIOMED-1 international cooperative group for diagnostics in hematology. It represented the

laboratory for molecular analysis centralization for the multicentric treatment protocols LAL

09/2000 and LAL 10/07 and performed the clonality assay and the MRD evaluation of all the

enrolled patients.

Our laboratory is part of the following international and national organizations:

-EuroMRD Consortium (http://www.euromrd.org/usr/pub/participants.php) that was established in

2001 (formerly known as European Study Group on MRD detection in ALL; ESG-MRD-ALL) and

consists of 43 MRD-PCR laboratories across 18 countries in Europe, Israel, Singapore, Japan and

Australia. This consortium collects only experienced laboratory in the field of TCR-Ig

rearrangements study for lymphomas and lymphoblastic leukemia (9) and produced protocols and

guidelines for MRD detection and data interpretation (6). It also organizes quality control rounds

every 6 month to monitor the performance of each laboratory and discuss the results in meetings

organized every year. This study group belongs to the ESLHO (European Scientific foundation for

Laboratory HematoOncology, http://www.eslho.org/usr/index.php ) and in collaboration with the

EuroClonality division (also belonging to ESLHO) is promoting a project for the validation of new

HTS based procedures for clonatity assessment and MRD evaluation.

- EuroMRD for Ph+ ALL which is working on guidelines for the quantitation of the BCR-ABL

minor transcript in B-cell acute lymphoblastic leukemia (13).

- LabNet, a National network for the molecular quantitation of the BCR-ABL major transcript in

Chronic Myeloid Leukemia according to LeukemiaNet recommendations (14) with an International

Scale Conversion Factor (CF) that is validated every year with quality control rounds.

Our laboratory is also a part of the international IRONII project aimed to evaluate the HTS utility in

the oncohaematologic field (15, 16). This working group is developing tools to identify the most

significant molecular alteration at diagnosis of the most common hematologic disease based on the

454 HTS technology. In particular, we belongs to the ALL working group in which a number of

significant molecular markers have been explored, among them p53 mutations were searched at

disease onset and the results are under evaluation.



Access to the instrumentations and facilities

Sequenta is a Company that provides clonality study and MRD evaluation on clinical samples with

HTS technology upon request. Furthermore, in our Institution are present two HTS platforms: the

MiSeq platform (Illumina) and the 454 Junior platform (Roche). A second MiSeq platform will be

available at later at the end of the year. All the instruments are accessible to research units and

bioinformatics support is guarantee.

Support

As above mentioned, our laboratory is part of the EuroMRD study which collects all the

experienced laboratory all around the Europe in the field of TCR-Ig rearrangements study for

lymphomas and lymphoblastic leukemia (9). The EuroMRD and EuroClonality working group have

already started collaborative efforts to validate the HTS in clonal rearrangements identification and

MRD evaluation.

Preliminary data

In 2000 in our hospital a multicenter treatment study for ALL (NILG trial 09/00, ClinicalTrials.gov

Id: NCT00358072) started to test prospectively the predictive value of minimal residual disease

(MRD) in adult ALL patients (n=280) (3). Our laboratory studied all the enrolled patients to

develop a molecular probe based on clone-specific T cell Receptor (TCR) or Immunoglobulin (Ig)

rearrangements. With this approach we were able to categorize patients in two distinct groups after

the consolidation therapy: one in which the minimal residual disease was undetectable (MRDneg,

n=58) and one with detectable residual disease (MRDpos, n=54); the first group of patient received

only chemotherapy whether the second group (n=54) was switched to an allogeneic stem cell

transplantation, when possible or an high dose chemotherapy course supported by an autologous

stem cell transplantation. The MRDneg group, treated with a two year chemotherapy, had a DFS of

72% weather the MRDpos group, treated with allogeneic transplantation or with high dose

chemotherapy and autologous transplantation, based on donor availability, had a DFS of 14% at 5

years (3). Patients in which it was not possible to generate a suitable molecular probe for MRD

monitoring (n=30) could not benefit of MRD categorization for subsequent treatment allocation.

These patients were treated according to clinical risk class and showed a DFS at 5 years of 40%.

This latter group would clearly benefit from a technology able to identify patients with no residual

disease already cured by chemotherapy and patients with persistence of disease in which therapy

intensification is recommended.



References

1. Bassan R, Hoelzer D. Modern therapy of acute lymphoblastic leukemia. J Clin Oncol

2011;29(5):532-43.

2. Pui CH, Carroll WL, Meshinchi S, Arceci RJ. Biology, risk stratification, and therapy of

pediatric acute leukemias: an update. J Clin Oncol 2011;29(5):551-65.

3. Bassan R, Spinelli O, Oldani E, Intermesoli T, Tosi M, Peruta B, et al. Improved risk

classification for risk-specific therapy based on the molecular study of minimal residual disease

(MRD) in adult acute lymphoblastic leukemia (ALL). Blood 2009;113(18):4153-62.

4. Bruggemann M, Raff T, Flohr T, Gokbuget N, Nakao M, Droese J, et al. Clinical

significance of minimal residual disease quantification in adult patients with standard-risk acute

lymphoblastic leukemia. Blood 2006;107(3):1116-23.

5. Flohr T, Schrauder A, Cazzaniga G, Panzer-Grumayer R, van der Velden V, Fischer S, et al.

Minimal residual disease-directed risk stratification using real-time quantitative PCR analysis of

immunoglobulin and T-cell receptor gene rearrangements in the international multicenter trial

AIEOP-BFM ALL 2000 for childhood acute lymphoblastic leukemia. Leukemia 2008;22(4):771-

82.

6. van der Velden VH, Cazzaniga G, Schrauder A, Hancock J, Bader P, Panzer-Grumayer ER,

et al. Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for

interpretation of real-time quantitative PCR data. Leukemia 2007;21(4):604-11.

7. Faham M, Zheng J, Moorhead M, Carlton VE, Stow P, Coustan-Smith E, et al. Deep-

sequencing approach for minimal residual disease detection in acute lymphoblastic leukemia. Blood

2012;120(26):5173-80.

8. Ladetto M, Bruggemann M, Monitillo L, Ferrero S, Pepin F, Drandi D, et al. Next-

generation sequencing and real-time quantitative PCR for minimal residual disease detection in B-

cell disorders. Leukemia 2013.

9. Bruggemann M, Schrauder A, Raff T, Pfeifer H, Dworzak M, Ottmann OG, et al.

Standardized MRD quantification in European ALL trials: proceedings of the Second International

Symposium on MRD assessment in Kiel, Germany, 18-20 September 2008. Leukemia

2010;24(3):521-35.

10. Szczepanski T, Willemse MJ, van Wering ER, van Weerden JF, Kamps WA, van Dongen

JJ. Precursor-B-ALL with D(H)-J(H) gene rearrangements have an immature immunogenotype with

a high frequency of oligoclonality and hyperdiploidy of chromosome 14. Leukemia

2001;15(9):1415-23.

11. van Dongen JJ, Macintyre EA, Gabert JA, Delabesse E, Rossi V, Saglio G, et al.

Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute

leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action:

investigation of minimal residual disease in acute leukemia. Leukemia 1999;13(12):1901-28.

12. Gabert J, Beillard E, van der Velden VHJ, Bi W, Grimwade D, Pallisgaard N, et al.

Standardization and quality control studies of /`real-time/' quantitative reverse transcriptase

polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - A

Europe Against Cancer Program. 2003;17(12):2318-2357.

13. Pfeifer HM, Giovanni Cazzaniga, Orietta Spinelli, Jean-Michel Cayuela, Hélène Cavé, Peter

Vandenberghe, MD, Ph, Carmen Chillon*, Tomasz Sacha, MD, Sandrine Hayette, PhD, Silja

Roettgers, PhD, Thomas Lion, MD, PhD, Professor, Letizia Foroni, MD PhD, Vincent H.J. van der

Velden, PhD, Jan Zuna, Monica Hermanson, PhD, Martin Christian Mueller, MD, Thoralf Lange,

Miroslaw Majewski, MD, Israel Bendit, MD, Fabrizio Pane, Ilaria Iacobucci, PhD, Veli Kairisto,

MD, PhD, Christa Homburg, Smadar Avigad, Eng Juh Allen Yeoh, Beat Schäfer, Adele Fielding,

MB BS, PhD, Loredana Elia, Katarzyna Borg, Eric Delabesse, Susanne Schnittger, PhD, Sandra

Markovic, Volker Werner, Nicola Goekbuget, MD, Dieter Hoelzer, MD PhD, Jacques J.M. van

Dongen, PhDand Oliver G. Ottmann, MD, PhD. International Standardization of Minimal Residual



Disease Assessment for in Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

(Ph+ALL) Expressing m-BCR-ABL Transcripts: Updated Results of Quality Control Procedures by

the EWALL and ESG-MRD-ALL Consortia. Blood 2011.

14. Baccarani M, Deininger MW, Rosti G, Hochhaus A, Soverini S, Apperley JF, et al.

European LeukemiaNet recommendations for the management of chronic myeloid leukemia: 2013.

Blood 2013;122(6):872-84.

15. Kohlmann A, Martinelli G, Hofmann W-K, Kronnie G, Chiaretti S, Preudhomme C, et al.

The Interlaboratory Robustness of Next-Generation Sequencing (IRON) Study Phase II: Deep-

Sequencing Analyses of Hematological Malignancies Performed by an International Network

Involving 26 Laboratories. ASH Annual Meeting Abstracts 2012;120(21):1399-.

16. Kohlmann A, Klein HU, Weissmann S, Bresolin S, Chaplin T, Cuppens H, et al. The

Interlaboratory RObustness of Next-generation sequencing (IRON) study: a deep sequencing

investigation of TET2, CBL and KRAS mutations by an international consortium involving 10

laboratories. Leukemia 2011;25(12):1840-8.



PERSONNEL INVOLVED IN THE RESEARCH Core Team Members

Supporting documentation available in:

Addendum A - PERSONNEL INVOLVED IN THE RESEARCH - CURRICULUM VITAE

Name Date of birth Institution Role on project Clinician Financial support/yr Man/Year effort

Doctor Alessandro Rambaldi 22/08/1955 Azienda Ospedaliera Papa GiovanniXXIII Principal Investigator Y N 0,20

Doctor Peruta Barbara 20/02/1981 Azienda Ospedaliera Papa GiovanniXXIII Fellow N € 22.000,00 1,00

Doctor Tosi Manuela 01/10/1975 Azienda Ospedaliera Papa GiovanniXXIII Fellow N N 1,00

Doctor Spinelli Orietta 04/03/1968 Azienda Ospedaliera Papa GiovanniXXIII Internal Collaborator N N 0,30

Borleri Gianmaria 22/09/1959 Azienda Ospedaliera Papa GiovanniXXIII Technician N N 0,20

Doctor Intermesoli Tamara 15/01/1975 Azienda Ospedaliera Papa GiovanniXXIII Internal Collaborator Y N 0,20

Total Man/Year on project 2,9

PERSONNEL INVOLVED IN THE RESEARCHRambaldi Alessandro [PI]


Description of work of each personnel

Rambaldi Alessandro, MD Head of Hematology and Bone Marrow Transplant Unit, is the

principal investigator of acute leukemia clinical trials active in the Unit.

Intermesoli Tamara, MD Senior consultant, is full time involved in management of patients

enrolled in clinical studies for acute and chronic leukemia.

Spinelli Orietta, PhD Senior Investigator, is full time involved in molecular study of minimal

residual disease in ALL patients. She will coordinate the sample analysis of selected patients to be

studied with HTS technology.

Manuela Tosi, PhD. Research fellow, involved in molecular study of minimal residual disease in

ALL patients.

Barbara Peruta, Biol. Sci., Research fellow will be involved in the following tasks:

Analysis of the diagnostic material of the patients enrolled into the clinical trials for clonal

rearrangements identification and subsequent MRD analysis.

Preparation and quality evaluation of samples for HTS procedure.

Analysis of the output sequences, identification of clonal markers and MRD levels.

Correlation of molecular results to clinical outcome of patients

Gianmaria Borleri, Senior technician is full time involved in the diagnostic workup (morphology,

flow cytometry and cell preparation of acute leukemia samples)

A schematic representation of the activity addressed to the various tasks is shown below:

Organizational chart

A.R. P.I.: Coordination and supervision of the research throughout the project

T.I. Coordination of activities related to clinical trials

O.S. Coordination of activities related to minimal residual disease analysis

M.T.

B.P.

G.B.

Analysis with HTS technique

Sequencing on the HTS platform

Analysis of the output sequences to identify Ig/TCR gene rearrangements

Comparison between Ig/TCR gene rearrangements identified by the new

HTS platform and by the conventional EuroMRD approach.

HTS sequencing of the end of consolidation samples of the same10 patients

and comparison of MRD results with those obtained with conventional AOS-

QPCR.

HTS analysis of the MRD unknown cohort of patients

Sequencing of MRDunk cohort of patients on the HTS platform

Analysis of the output sequences to identify Ig/TCR gene rearrangements

Outcome analysis of MRDunk cohort newly re-classified as MRDpos or

MRDneg.

Prospective application of the HTS technique in parallel with conventional method

on newly diagnosed ALL patients

Prospective Ig/TCR gene rearrangements identification with new HTS

technique.

Analysis of MRD on samples collected at the end of consolidation therapy

with the new HTS method.

Evaluation of feasibility and applicability of this analysis in a prospective

clinical trial in which treatment are decided based on MRD results.

DESCRIPTION OF THE WORK FOR EVERY UNIT OF PERSONNELRambaldi Alessandro [PI]


BUDGET FORM AND JUSTIFICATIONS

1st year 2nd year 3rd year Total

Direct Research costs

- Consumables and supplies € 40.000,00 € 40.000,00 € 39.000,00 € 119.000,00

- Small bench instrumentation € 0,00 € 0,00 € 0,00 € 0,00

- Services € 5.000,00 € 5.000,00 € 5.000,00 € 15.000,00

- Maintenance contracts € 0,00 € 0,00 € 0,00 € 0,00

- Publication costs € 0,00 € 0,00 € 1.000,00 € 1.000,00

- Meetings and travel costs € 1.000,00 € 1.000,00 € 1.000,00 € 3.000,00

Personnel costs € 22.000,00 € 22.000,00 € 22.000,00 € 66.000,00

Indirect costs (9,5%) € 6.460,00 € 6.460,00 € 6.460,00 € 19.380,00

SUBTOTAL € 74.460,00 € 74.460,00 € 74.460,00 € 223.380,00

Overheads (10,0%) € 7.446,00 € 7.446,00 € 7.446,00 € 22.338,00

Total € 81.906,00 € 81.906,00 € 81.906,00 € 245.718,00

Proponent's signature Legal Representative's signature Date_____________________ _____________________ 18 mar 2014Rambaldi Alessandro Nicora Carlo

BUDGET FORM AND JUSTIFICATIONSRambaldi Alessandro [PI]


CONSUMABLES AND SUPPLIES

High Throughput Sequencing kit and reagents, disposable plasticware, ficoll, DNA extraction kits, antibodies for flowcytometry analysis,

ASO-QPCR reagents.Chips for DNA quality evaluation with Bioanalyzer

SMALL BENCH INSTRUMENTATION

not requested

SERVICES

service for allele specific oligonucleotide synthesis

MAINTENANCE CONTRACTS

not requested

PUBLICATIONS COSTS

estimated cost for publication and/or abstract submission at meetings with fee, including expenses for poster printing for the last year project

MEETING AND TRAVEL COSTS

Estimated costs for two young fellow participation to meeting and congresses related to the project

PERSONNEL COSTS

Annual estimated cost for research fellowship involved in the following tasks:

• Analysis of the diagnostic material of the patients enrolled into the clinical trials for clonal rearrangements identification and subsequent

MRD analysis.

• Preparation and quality evaluation of samples for HTS procedure.

• Analysis of the output sequences, identification of clonal markers and MRD levels.

• Correlation of molecular results to clinical outcome of patients

Letter of Indirect Costs/Overheads available in:

Addendum B - BUDGET FORM AND JUSTIFICATIONS

BUDGET FORM AND JUSTIFICATIONSRambaldi Alessandro [PI]


BIOGRAPHICAL SKETCH

PERSONAL DATA OF THE PI

SurnameRambaldi

NameAlessandro

PositionHead

Date of birth22/08/1955

EDUCATION AND TRAINING (INCLUDES DEGREES AND POST-DOCTORAL TRAINING)

Duration(from/to) Degree/Training Field of study Institution City

Oct 1987/Nov 1991 Specialist Hematology Università degli Studi di Bari

Aldo Moro Bari

Oct 1982/Jul 1985 Specialist Clinical Pharmacology Università degli Studi di

Milano Milan

Sep 1975/Nov 1981 Medical degree Medicine Università degli Studi di

Milano Milan

RESEARCH AND PROFESSIONAL EXPERIENCE

Duration(from/to) Position Institution City Country

Jan 2006/Mar 2014

Head of Hematology and BoneMarrow Transplant Unit

Azienda OspedalieraPapa Giovanni XXIII Bergamo Italy

Dec 1999/Dec 2006

Head of Bone Marrow TransplantProgram

Azienda OspedalieraOspedali Riuniti di

BergamoBergamo Italy

Dec 1994/Dec 1999 Senior Consultant in Hematology

Azienda OspedalieraOspedali Rinuti di


Jan 1991/Dec 1994

Head Unit of Molecular Biology andBlood Diseases

Istituto di RicercheFarmacologiche "Mario

Negri" I.R.C.C.S.Milano Italy

Dec 1989/Dec 1994 Physician staff, Hematology Division

Azienda OspedalieraOspedali Riuniti di


Jan 1988/Dec 1989 Senior Scientist



Jan 1986/Dec 1987 Research associate

Dana Farber CancerInstitute, HarvardMedical School

Boston United States ofAmerica

Jan 1982/Dec 1985 Research associate



BIOGRAPHICAL SKETCHRambaldi Alessandro [PI]


Rambaldi’s scientific achievements

1) Development of innovative strategies for the molecular diagnosis and

monitoring of genetic abnormalities in hematologic malignancies.

2) Pioneering the clinical development of Rituximab in Follicular non Hodgkin

Lymphoma.

3) Pioneering risk adapted treatment strategy in adult ALL. This approach based

on a real time evaluation of molecular minimal residual disease has become a

standard of care for this disease.

4) Introducing new cellular therapies in patients relapsing after allogeneic stem

cell transplantation.

NARRATIVE BIOSKETCHRambaldi Alessandro [PI]


PI TRACK RECORD SUMMARY (2009-2014)

This track record summary is based on the publications by the PI in the last five years, listed in the following page(s). The summary does not include Papers in press. The applicant certifies that all publications have been carefully checked and correctly flagged for authorship.

Principal Investigator's Full NameRambaldi Alessandro

Total number of papers 46

Total number of papers with IF 46

Total IF 342,467

Average IF (of papers with IF) 7,4

Number of papers as First, Last, or Corresponding Author (all journals) 13

Number of papers as First, Last, or Corresponding Author (journals with IF) 13

Active IF 89,002

Average Active IF 6,8

Number of papers relevant for cancer research 45

Number of papers with acknowledgement to AIRC 26

PI TRACK RECORD SUMMARYRambaldi Alessandro [PI]


PUBLICATIONS OF THE PI Title, list of authors and complete reference of all publications from the last 5 years (2009 - 2014)

FA/CoF = First Author/Co-First Author

LA/CoL/CA/CC = Last Author/Co-Last Author/Corresponding Author/Co-Corresponding Author

Principal Investigator's Full Name: Rambaldi Alessandro

Publication IF FA/CoF LA/CoL/CA/CC Relev. Ackn.

Allogenic hematopoietic stem cell transplantation in patients with polycythemia or essential thrombocythemia transformed to myelofibrosisor acute myeloid leukemia: report from the MPN subcommittee of the Chronic Malignancies Working Party of the European Group forBlood and Marrow Transplantation.

Lussana F, Rambaldi A, Finazzi MC, van Biezen A, Scholten M, Oldani E, Carobbio A, Iacobelli S, Finke J, Nagler A, Volin L, Lamy T,Arnold R, Mohty M, Michallet M, De Witte T, Olavarria E, Kröger N

HAEMATOL-HEMATOL J 2014 Feb; :

5,935 X

Discriminating between essential thrombocythemia and masked polycythemia vera in JAK2 mutated patients.

Barbui T, Thiele J, Carobbio A, Guglielmelli P, Rambaldi A, Vannucchi AM, Tefferi A

AM J HEMATOL 2014 Feb; :

4,138 X AIRC

Distinct clustering of symptomatic burden amongst myeloproliferative neoplasm patients: retrospective assessment in 1470 patients.

Geyer HL, Emanuel RM, Dueck AC, Kiladjian JJ, Xiao Z, Slot S, Zweegman S, Sackmann F, Kerguelen Fuentes A, Hernández-Maraver D,Döhner K, Harrison CN, Radia D, Muxi P, Besses C, Cervantes F, Johansson PL, Andreasson B, Rambaldi A, Barbui T, Vannucchi AM,Passamonti F, Samuelsson J, Birgegard G, Mesa RA

BLOOD 2014 Feb; :

9,06 X

Highly aggressive T-cell acute lymphoblastic leukemia with t(8;14)(q24;q11): extensive genetic characterization and achievement of earlymolecular remission and long-term survival in an adult patient.

Parolini M, Mecucci C, Matteucci C, Giussani U, Intermesoli T, Tosi M, Rambaldi A, Bassan R

BLOOD CANCER J 2014; 4: e176

1,4 X

Predictive factors for the outcome of allogeneic transplantation in patients with myelodysplastic syndrome stratified according to the revisedInternational Prognostic Scoring System (IPSS-R).

Della Porta MG, Alessandrino EP, Bacigalupo A, van Lint MT, Malcovati L, Pascutto C, Falda M, Bernardi M, Onida F, Guidi S, Iori AP,Cerretti R, Marenco P, Pioltelli P, Angelucci E, Oneto R, Ripamonti F, Bernasconi P, Bosi A, Cazzola M, Rambaldi A

BLOOD 2014 Feb; :

9,06 X X AIRC

PUBLICATIONS OF THE PIRambaldi Alessandro [PI]


Results of a randomized trial comparing high-dose chemotherapy plus Auto-SCT and R-FC in CLL at diagnosis.

Magni M, Nicola MD, Patti C, Scimè R, Mulè A, Rambaldi A, Intermesoli T, Viero P, Tarella C, Gueli A, Bergui L, Trentin L, Barzan A,Benedetti F, Ambrosetti A, Di Raimondo F, Chiarenza A, Parvis G, Billio A, Attolico I, Olivieri A, Montanari M, Carlo-Stella C, MatteucciP, Devizzi L, Guidetti A, Viviani S, Valagussa P, Gianni AM

BONE MARROW TRANSPL 2014 Jan; :

3,541 X

Treatment of Graft versus Host Disease with Mesenchymal Stromal Cells: A Phase I Study on 40 Adult and Pediatric Patients.

Introna M, Lucchini G, Dander E, Galimberti S, Rovelli A, Balduzzi A, Longoni D, Pavan F, Masciocchi F, Algarotti A, Micò C, Grassi A,Deola S, Cavattoni I, Gaipa G, Belotti D, Perseghin P, Parma M, Pogliani E, Golay J, Pedrini O, Capelli C, Cortelazzo S, D'Amico G, BiondiA, Rambaldi A, Biagi E

BIOL BLOOD MARROW TR 2014 Mar; 20: 375-81

3,94 X AIRC

A phase 2 study of ruxolitinib, an oral JAK1 and JAK2 inhibitor, in patients with advanced polycythemia vera who are refractory orintolerant to hydroxyurea.

Verstovsek S, Passamonti F, Rambaldi A, Barosi G, Rosen PJ, Rumi E, Gattoni E, Pieri L, Guglielmelli P, Elena C, He S, Contel N,Mookerjee B, Sandor V, Cazzola M, Kantarjian HM, Barbui T, Vannucchi AM

CANCER-AM CANCER SOC 2013 Oct; :

5,201

Cytokine Induced Killer (CIK) cells for the treatment of haematological neoplasms.

Introna M, Golay J, Rambaldi A

IMMUNOL LETT 2013 Sep-Oct; 155: 27-30

2,337 X X AIRC

Elevated C-reactive protein is associated with shortened leukemia-free survival in patients with myelofibrosis.

Barbui T, Carobbio A, Finazzi G, Guglielmelli P, Salmoiraghi S, Rosti V, Rambaldi A, Vannucchi AM, Barosi G

LEUKEMIA 2013 Oct; 27: 2084-6

10,164 X AIRC

High cure rates in Burkitt lymphoma and leukemia: a Northern Italy Leukemia Group study of the German short intensive rituximab-chemotherapy program.

Intermesoli T, Rambaldi A, Rossi G, Delaini F, Romani C, Pogliani EM, Pagani C, Angelucci E, Terruzzi E, Levis A, Cassibba V, Mattei D,Gianfaldoni G, Scattolin AM, Di Bona E, Oldani E, Parolini M, Gökbuget N, Bassan R

HAEMATOL-HEMATOL J 2013 Nov; 98: 1718-25

5,935 X

Mutations and chromosomal rearrangements of JAK2: not only a myeloid issue.

Salmoiraghi S, Montalvo ML, D'Agostini E, Amicarelli G, Minnucci G, Spinelli O, Rambaldi A

EXPERT REV HEMATOL 2013 Aug; 6: 429-39

2,382 X X AIRC



Retinoic acid and arsenic trioxide for acute promyelocytic leukemia.

Lo-Coco F, Avvisati G, Vignetti M, Thiede C, Orlando SM, Iacobelli S, Ferrara F, Fazi P, Cicconi L, Di Bona E, Specchia G, Sica S, DivonaM, Levis A, Fiedler W, Cerqui E, Breccia M, Fioritoni G, Salih HR, Cazzola M, Melillo L, Carella AM, Brandts CH, Morra E, vonLilienfeld-Toal M, Hertenstein B, Wattad M, Lübbert M, Hänel M, Schmitz N, Link H, Kropp MG, Rambaldi A, La Nasa G, Luppi M, CiceriF, Finizio O, Venditti A, Fabbiano F, Döhner K, Sauer M, Ganser A, Amadori S, Mandelli F, Döhner H, Ehninger G, Schlenk RF,Platzbecker U, Gruppo Italiano Malattie Ematologiche dell'Adulto, German-Austrian Acute Myeloid Leukemia Study Group, Study AllianceLeukemia

NEW ENGL J MED 2013 Jul; 369: 111-21

51,658 X AIRC

Telomere shortening in Ph-negative chronic myeloproliferative neoplasms: A biological marker of polycythemia vera and myelofibrosis,regardless ofhydroxycarbamide therapy.

Ruella M, Salmoiraghi S, Risso A, Carobbio A, Buttiglieri S, Spatola T, Sivera P, Ricca I, Barbui T, Tarella C, Rambaldi A

EXP HEMATOL 2013 Jul; 41: 627-34

2,907 X X AIRC

The CIBMTR score predicts survival of AML patients undergoing allogeneic transplantation with active disease after a myeloablative orreduced intensity conditioning: a retrospective analysis of the Gruppo Italiano Trapianto Di Midollo Osseo.

Todisco E, Ciceri F, Oldani E, Boschini C, Micò C, Vanlint MT, Donnini I, Patriarca F, Alessandrino PE, Bonifazi F, Arcese W, Barberi W,Marenco P, Terruzzi E, Cortelazzo S, Santarone S, Proia A, Corradini P, Tagliaferri E, Falcioni S, Irrera G, Dallanegra L, Castagna L,Santoro A, Camboni A, Sacchi N, Bosi A, Bacigalupo A, Rambaldi A

LEUKEMIA 2013 Oct; 27: 2086-91

10,164 X X AIRC

The lymphocyte to monocyte ratio improves the IPI-risk definition of diffuse large B-cell lymphoma when rituximab is added tochemotherapy.

Rambaldi A, Boschini C, Gritti G, Delaini F, Oldani E, Rossi A, Barbui AM, Caracciolo D, Ladetto M, Gueli A, De Crescenzo A, Passera R,Devizzi L, Patti C, Gianni AM, Tarella C

AM J HEMATOL 2013 Dec; 88: 1062-7

4,138 X X AIRC

A novel murine model of myeloproliferative disorders generated by overexpression of the transcription factor NF-E2.

Kaufmann KB,Gründer A,Hadlich T,Wehrle J,Gothwal M,Bogeska R,Seeger TS,Kayser S,Pham KB,Jutzi JS,Ganzenmüller L,SteinemannD,Schlegelberger B,Wagner JM,Jung M,Will B,Steidl U,Aumann K,Werner M,Günther T,Schüle R,Rambaldi A,Pahl HL

J EXP MED 2012 Jan; 209: 35-50

13,214 X

A novel, highly sensitive and rapid Allele Specific-Loop mediated Amplification assay for the detection of the JAK2V617F mutation inchronic myeloproliferative neoplasms.

Minnucci G,Amicarelli G,Salmoiraghi S,Spinelli O,Guinea Montalvo ML,Giussani U,Adlerstein D,Rambaldi A

HAEMATOL-HEMATOL J 2012 Sep; 97: 1394-400

5,935 X X AIRC

ASXL1 mutations in primary and secondary myelofibrosis.

Ricci C,Spinelli O,Salmoiraghi S,Finazzi G,Carobbio A,Rambaldi A

BRIT J HAEMATOL 2012 Feb; 156: 404-7

4,942 X X AIRC



CD20 expression has no prognostic role in Philadelphia-negative B-precursor acute lymphoblastic leukemia: new insights from the molecularstudy of minimal residual disease.

Mannelli F, Gianfaldoni G, Intermesoli T, Cattaneo C, Borlenghi E, Cortelazzo S, Cavattoni I, Pogliani EM, Fumagalli M, Angelucci E,Romani C, Ciceri F, Corti C, Scattolin A, Cortelezzi A, Mattei D, Audisio E, Spinelli O, Oldani E, Bosi A, Rambaldi A, Bassan R

HAEMATOL-HEMATOL J 2012 Apr; 97: 568-71

5,935 X

Chronic GVHD is associated with lower relapse risk irrespective of stem cell source among patients receiving transplantation from unrelateddonors.

Signori A, Crocchiolo R, Oneto R, Sacchi N, Sormani MP, Fagioli F, Rambaldi A, Ciceri F, Bacigalupo A

BONE MARROW TRANSPL 2012 Nov; 47: 1474-8

3,541 X

Mesenchymal stromal cells for the treatment of graft-versus-host disease: understanding the in vivo biological effect through patient immunemonitoring.

Dander E, Lucchini G, Vinci P, Introna M, Masciocchi F, Perseghin P, Balduzzi A, Bonanomi S, Longoni D, Gaipa G, Belotti D, Parma M,Algarotti A, Capelli C, Golay J, Rovelli A, Rambaldi A, Biondi A, Biagi E, D'Amico G

LEUKEMIA 2012 Jul; 26: 1681-4

10,164 X

Outcome of patients activating an unrelated donor search: the impact of transplant with reduced intensity conditioning in a large cohort ofconsecutive high-risk patients.

Rambaldi A,Bacigalupo A,Fanin R,Ciceri F,Bonifazi F,Falda M,Lambertenghi-Deliliers G,Benedetti F,Bruno B,Corradini P,AlessandrinoPE,Iacopino P,Arcese W,Scimè R,Raimondi R,Sica S,Castagna L,Lamparelli T,Oneto R,Lombardini L,Pollichieni S,Algarotti A,CarobbioA,Sacchi N,Bosi A

LEUKEMIA 2012 Aug; 26: 1779-85

10,164 X X AIRC

p38aMAPK interacts with and inhibits RARa: suppression of the kinase enhances the therapeutic activity of retinoids in acute myeloidleukemia cells.

Gianni M, Peviani M, Bruck N, Rambaldi A, Borleri G, Terao M, Kurosaki M, Paroni G, Rochette-Egly C, Garattini E

LEUKEMIA 2012 Aug; 26: 1850-61

10,164 X

Results of a lymphoblastic leukemia-like chemotherapy program with risk-adapted mediastinal irradiation and stem cell transplantation foradult patients with lymphoblastic lymphoma.

Cortelazzo S, Intermesoli T, Oldani E, Ciceri F, Rossi G, Pogliani EM, Mattei D, Romani C, Cortelezzi A, Borlenghi E, Corti C, Peruta B,Spinelli O, Rambaldi A, Bassan R

ANN HEMATOL 2012 Jan; 91: 73-82

2,866 X AIRC

The HDAC Inhibitor Givinostat Modulates the Hematopoietic Transcription Factors NFE2 and C-MYB in JAK2V617F MyeloproliferativeNeoplasm Cells.

Calzada AA,Todoerti K,Donadoni L,Pellicioli A,Tuana G,Gatta R,Neri A,Finazzi G,Mantovani R,Rambaldi A,Introna M,Lombardi L,GolayJ

EXP HEMATOL 2012 Aug; 40: 634-45.e10

2,907 X AIRC



AIDA 0493 protocol for newly diagnosed acute promyelocytic leukemia: very long-term results and role of maintenance.

Avvisati G, Lo-Coco F, Paoloni FP, Petti MC, Diverio D, Vignetti M, Latagliata R, Specchia G, Baccarani M, Di Bona E, Fioritoni G,Marmont F, Rambaldi A, Di Raimondo F, Kropp MG, Pizzolo G, Pogliani EM, Rossi G, Cantore N, Nobile F, Gabbas A, Ferrara F, Fazi P,Amadori S, Mandelli F, GIMEMA, AIEOP, and EORTC Cooperative Groups

BLOOD 2011 May; 117: 4716-25

9,06 X

Dual-functional capability of CD3+CD56+ CIK cells, a T-cell subset that acquires NK function and retains TCR-mediated specificcytotoxicity.

Pievani A, Borleri G, Pende D, Moretta L, Rambaldi A, Golay J, Introna M

BLOOD 2011 Sep; 118: 3301-10

9,06 X AIRC

Enhanced killing of human B-cell lymphoma targets by combined use of cytokine-induced killer cell (CIK) cultures and anti-CD20antibodies.

Pievani A, Belussi C, Klein C, Rambaldi A, Golay J, Introna M

BLOOD 2011 Jan; 117: 510-8

9,06 X AIRC

Inflammation and thrombosis in essential thrombocythemia and polycythemia vera: different role of C-reactive protein and pentraxin 3.

Barbui T, Carobbio A, Finazzi G, Vannucchi AM, Barosi G, Antonioli E, Guglielmelli P, Pancrazzi A, Salmoiraghi S, Zilio P, Ottomano C,Marchioli R, Cuccovillo I, Bottazzi B, Mantovani A, Rambaldi A, AGIMM and IIC Investigators

HAEMATOL-HEMATOL J 2011 Feb; 96: 315-8

5,935 X X AIRC

Mechanism of action of type II, glycoengineered, anti-CD20 monoclonal antibody GA101 in B-chronic lymphocytic leukemia whole bloodassays in comparison with rituximab and alemtuzumab.

Bologna L, Gotti E, Manganini M, Rambaldi A, Intermesoli T, Introna M, Golay J

J IMMUNOL 2011 Mar; 186: 3762-9

5,52 X AIRC

Mutations of CD79A, CD79B and EZH2 genes in immunodeficiency-related non-Hodgkin lymphomas.

Capello D, Gloghini A, Martini M, Spina M, Tirelli U, Bertoni F, Rinaldi A, Morra E, Rambaldi A, Sinigaglia F, Larocca LM, Carbone A

BRIT J HAEMATOL 2011 Mar; 152: 777-80

4,942 X

Whole-exome sequencing identifies somatic mutations of BCOR in acute myeloid leukemia with normal karyotype.

Grossmann V, Tiacci E, Holmes AB, Kohlmann A, Martelli MP, Kern W, Spanhol-Rosseto A, Klein HU, Dugas M, Schindela S, TrifonovV, Schnittger S, Haferlach C, Bassan R, Wells VA, Spinelli O, Chan J, Rossi R, Baldoni S, De Carolis L, Goetze K, Serve H, Peceny R,Kreuzer KA, Oruzio D, Specchia G, Di Raimondo F, Fabbiano F, Sborgia M, Liso A, Farinelli L, Rambaldi A, Pasqualucci L, Rabadan R,Haferlach T, Falini B

BLOOD 2011 Dec; 118: 6153-63

9,06 X AIRC



Chemotherapy-phased imatinib pulses improve long-term outcome of adult patients with Philadelphia chromosome-positive acutelymphoblastic leukemia: Northern Italy Leukemia Group protocol 09/00.

Bassan R, Rossi G, Pogliani EM, Di Bona E, Angelucci E, Cavattoni I, Lambertenghi-Deliliers G, Mannelli F, Levis A, Ciceri F, Mattei D,Borlenghi E, Terruzzi E, Borghero C, Romani C, Spinelli O, Tosi M, Oldani E, Intermesoli T, Rambaldi A

J CLIN ONCOL 2010 Aug; 28: 3644-52

18,038 X X AIRC

Feasibility and safety of adoptive immunotherapy with CIK cells after cord blood transplantation.

Introna M, Pievani A, Borleri G, Capelli C, Algarotti A, Micò C, Grassi A, Oldani E, Golay J, Rambaldi A

BIOL BLOOD MARROW TR 2010 Nov; 16: 1603-7

3,94 X X AIRC

Pleiotropic anti-myeloma activity of ITF2357: inhibition of interleukin-6 receptor signaling and repression of miR-19a and miR-19b.

Todoerti K, Barbui V, Pedrini O, Lionetti M, Fossati G, Mascagni P, Rambaldi A, Neri A, Introna M, Lombardi L, Golay J

HAEMATOL-HEMATOL J 2010 Feb; 95: 260-9

5,935 X AIRC

Single nucleotide polymorphism-arrays provide new insights in the pathogenesis of post-transplant diffuse large B-cell lymphoma.

Rinaldi A, Capello D, Scandurra M, Greiner TC, Chan WC, Bhagat G, Rossi D, Morra E, Paulli M, Rambaldi A, Rancoita PM, Inghirami G,Ponzoni M, Moreno SM, Piris MA, Mian M, Chigrinova E, Zucca E, Favera RD, Gaidano G, Kwee I, Bertoni F

BRIT J HAEMATOL 2010 May; 149: 569-77

4,942 X

Standardized MRD quantification in European ALL trials: proceedings of the Second International Symposium on MRD assessment in Kiel,Germany, 18-20 September 2008.

Brüggemann M, Schrauder A, Raff T, Pfeifer H, Dworzak M, Ottmann OG, Asnafi V, Baruchel A, Bassan R, Benoit Y, Biondi A, Cavé H,Dombret H, Fielding AK, Foà R, Gökbuget N, Goldstone AH, Goulden N, Henze G, Hoelzer D, Janka-Schaub GE, Macintyre EA, Pieters R,Rambaldi A, Ribera JM, Schmiegelow K, Spinelli O, Stary J, von Stackelberg A, Kneba M, Schrappe M, van Dongen JJ, European WorkingGroup for Adult Acute Lymphoblastic Leukemia (EWALL), International Berlin-Frankfurt-Münster Study Group (I-BFM-SG)

LEUKEMIA 2010 Mar; 24: 521-35

10,164 X

Therapeutic efficacy of the pan-cdk inhibitor PHA-793887 in vitro and in vivo in engraftment and high-burden leukemia models.

Alzani R, Pedrini O, Albanese C, Ceruti R, Casolaro A, Patton V, Colotta F, Rambaldi A, Introna M, Pesenti E, Ciomei M, Golay J

EXP HEMATOL 2010 Apr; 38: 259-269.e2

2,907 X AIRC

A short low-dose imatinib trial allows rapid identification of responsive patients in hypereosinophilic syndromes.

Intermesoli T, Delaini F, Acerboni S, Salmoiraghi S, Spinelli O, Guerini V, Vannucchi AM, Mappa S, Rossi G, Rossi V, Di Bona E, ParatoreS, Carobbio A, Rambaldi A, Barbui T, Bassan R

BRIT J HAEMATOL 2009 Dec; 147: 681-5

4,942 X




Addendum C - PUBLICATIONS OF THE PI

HLA matching affects clinical outcome of adult patients undergoing haematopoietic SCT from unrelated donors: a study from the GruppoItaliano Trapianto di Midollo Osseo and Italian Bone Marrow Donor Registry.

Crocchiolo R, Ciceri F, Fleischhauer K, Oneto R, Bruno B, Pollichieni S, Sacchi N, Sormani MP, Fanin R, Bandini G, Bonifazi F, Bosi A,Rambaldi A, Alessandrino PE, Falda M, Bacigalupo A

BONE MARROW TRANSPL 2009 Nov; 44: 571-7

3,541 X

Identification of patients with poorer survival in primary myelofibrosis based on the burden of JAK2V617F mutated allele.

Guglielmelli P, Barosi G, Specchia G, Rambaldi A, Lo Coco F, Antonioli E, Pieri L, Pancrazzi A, Ponziani V, Delaini F, Longo G,Ammatuna E, Liso V, Bosi A, Barbui T, Vannucchi AM

BLOOD 2009 Aug; 114: 1477-83

9,06 X

Improved risk classification for risk-specific therapy based on the molecular study of minimal residual disease (MRD) in adult acutelymphoblastic leukemia (ALL).

Bassan R, Spinelli O, Oldani E, Intermesoli T, Tosi M, Peruta B, Rossi G, Borlenghi E, Pogliani EM, Terruzzi E, Fabris P, Cassibba V,Lambertenghi-Deliliers G, Cortelezzi A, Bosi A, Gianfaldoni G, Ciceri F, Bernardi M, Gallamini A, Mattei D, Di Bona E, Romani C,Scattolin AM, Barbui T, Rambaldi A

BLOOD 2009 Apr; 113: 4153-62

9,06 X X AIRC

Inhibition of the peptidyl-prolyl-isomerase Pin1 enhances the responses of acute myeloid leukemia cells to retinoic acid via stabilization ofRARalpha and PML-RARalpha.

Gianni' M, Boldetti A, Guarnaccia V, Rambaldi A, Parrella E, Raska, Rochette-Egly C, Del Sal G, Rustighi A, Terao M, Garattini E

CANCER RES 2009 Feb; 69: 1016-26

8,65 X AIRC

JAK2V617F allele burden and thrombosis: a direct comparison in essential thrombocythemia and polycythemia vera.

Carobbio A, Finazzi G, Antonioli E, Guglielmelli P, Vannucchi AM, Dellacasa CM, Salmoiraghi S, Delaini F, Rambaldi A, Barbui T

EXP HEMATOL 2009 Sep; 37: 1016-21

2,907 X

Molecular monitoring of residual disease in chronic myeloid leukemia by genomic DNA compared with conventional mRNA analysis.

Mattarucchi E, Spinelli O, Rambaldi A, Pasquali F, Lo Curto F, Campiotti L, Porta G

J MOL DIAGN 2009 Sep; 11: 482-7

3,952 X



BIO-ETHICAL REQUIREMENTS


Addendum D - BIO-ETHICAL REQUIREMENTS

Research on humans

Does the research plan include clinical trials with patients and/or healthy volunteers, or involve the use of human biologicalsamples, genetic material or data collection?

YES

NO

If yes, you MUST check one of the following boxes:

I have obtained the clearance from the competent Ethics Committee/Institutional Review Board, and I am attaching it tothe application, together with a copy of the informed consent if requested by the competent EthicsCommittee/Institutional Review Board.I have not obtained the clearance from the competent Ethics Committee/Institutional Review Board yet, but I haverequested it and will send it to the AIRC Peer Review Office by the deadline indicated in the Call, together with a copyof the informed consent if requested by the competent Ethics Committee/Institutional Review Board.

In any case, if the research deals with human biological samples, genetic material or data collection the applicant declares thatthe research plan includes information about:1) how the samples, material or data are collected;2) whether the samples, material or data are collected specifically for the proposed research project;3) how the samples, material or data are dismissed.

Proponent's signature Date_____________________ 18 mar 2014Rambaldi Alessandro

Research on animals

Does the proposed research involve animal experimentation?

YES

NO

If yes, you MUST check one of the following boxes:

I have obtained the clearance from the competent animal research ethics committee to carry out the described animalexperimentation, and I am attaching it to the application;I have not obtained the clearance from the competent animal research ethics committee yet, but I have requested it andwill send it to the AIRC Peer Review Office by the deadline indicated in the Call;there is not an active animal research ethics committee in my Institute, but the procedures related to animal use havebeen communicated to the Italian Ministry of Health and a copy of this communication is attached to the currentapplication;there is not an active animal research ethics committee in my Institute; I have yet to communicate the procedures relatedto animal use to the Italian Ministry of Health but I will do so and I will send a copy of this communication to the AIRCPeer Review Office by the deadline indicated in the Call.

In any case, the applicant declares that the research studies are accurately described in the proposal and conform to allregulations protecting animals used for research purposes, including those of the DL 116/92. The experiments described in theproposal will be performed following the guidelines described in: Workman P. et al.: 'Guidelines for the welfare and use ofanimals in cancer research'. Br. J. Cancer (2010) 102: 1555-1577.

In addition, the applicant declares that the principles of the three Rs (Replacement, Reduction, Refinement) have beeenimplemented in the research plan, as described in the attached PDF file.

Animal experimentation: Principles of the 3Rs

Proponent's signature Date_____________________ 18 mar 2014Rambaldi Alessandro

BIO-ETHICAL REQUIREMENTSRambaldi Alessandro [PI]


Personal Information

Name BARBARA PERUTA

Address

Phone number (+39) 0352673766

E-mail

Nationality Italian

Date and place of birth 20/02/1981

Educational Background

2005: Academic Degree in Medical Biotechnology with the full marks (110/110 cum laude) at the

University of Milan-Bicocca, Italy.

The title of the dissertation work was: “Adult acute lumphoblastic leukemia: prognostic impact of minimal

residual disease evaluation in allogeneic stem cells transplated patients”.

Positions and Employment

2013-2014 Fellow, Division of Molecular Hematology, A.O. “Papa Giovanni XXIII”, Bergamo, Italy

2006-2012 Fellow, Division of Molecular Hematology, Ospedali Riuniti di Bergamo, Italy

2004-2005 Student Fellow, Division of Molecular Hematology, Ospedali Riuniti di Bergamo, Italy

Other Experience and Professional Memberships:

2008-2014: member of the EUROMRD group. The EuroMRD Consortium was established in 2001 and

consists of 43 MRD-PCR laboratories across 18 countries in Europe, Israel, Singapore, Japan and Australia. The main aims of EuroMRD are:

1. Organisation of a quality-control program twice per year;

2. Collaborative development and evaluation of new MRD strategies and techniques;

3. Development of guidelines for the interpretation of RQ-PCR-based MRD data.

Work Experience:

Cell biology techniques: cell culture, immunohistochemistry, immunophenotyping (basic level), tumor

antigens targeting (basic level).

Molecular Biology: RNA and DNA extraction and preparation, RT-PCR, PCR and real time PCR, DNA

sequence, primer design, set up of multiplex fluorescence PCR, Genescan and fragment analysis

Specialist of molecular diagnosis of adult acute lymphoid Leukemias and lymphoblastic lymphomas and

molecular monitoring of minimal residual disease (MRD) in adult lymphoblastic diseases.

Publications

Cortelazzo S, Intermesoli T, Oldani E, Ciceri F, Rossi G, Pogliani EM, Mattei D, Romani C,

Cortelezzi A, Borlenghi E, Corti C, Peruta B, Spinelli O, Rambaldi A, Bassan R.

Results of a lymphoblastic leukemia-like chemotherapy program with risk-adapted mediastinal

irradiation and stem cell transplantation for adult patients with lymphoblastic lymphoma.

Ann Hematol. 2012 Jan;91(1):73-82.

Bassan R, Spinelli O, Oldani E, Intermesoli T, Tosi M, Peruta B, Rossi G, Borlenghi E, Pogliani

EM, Terruzzi E, Fabris P, Cassibba V, Lambertenghi-Deliliers G, Cortelezzi A, Bosi A, Gianfaldoni

G, Ciceri F, Bernardi M, Gallamini A, Mattei D, Di Bona E, Romani C, Scattolin AM, Barbui T,

Rambaldi A.

Improved risk classification for risk-specific therapy based on the molecular study of minimal

residual disease (MRD) in adult acute lymphoblastic leukemia (ALL).

Blood. 2009 Apr 30;113(18):4153-62.

Spinelli O, Peruta B, Tosi M, Guerini V, Salvi A, Zanotti MC, Oldani E, Grassi A, Intermesoli T,

Micò C, Rossi G, Fabris P, Lambertenghi-Deliliers G, Angelucci E, Barbui T, Bassan R, Rambaldi

A.

Clearance of minimal residual disease after allogeneic stem cell transplantation and the prediction of

the clinical outcome of adult patients with high-risk acute lymphoblastic leukemia.

Haematologica. 2007 May;92(5):612-8.

Addendum A - PERSONNEL INVOLVED IN THE RESEARCH - CURRICULUM VITAERambaldi Alessandro [PI]


Personal Information

Name TOSI MANUELA

Address

Phone number (+39) 0352673766

E-mail [email protected]

Nationality Italian

Date and place of birth 01/10/1975

Educational Background

2005 Postgraduate in Clinical Biochemistry

2000 Academic degree in Biological Sciences

Research experience

1999-2000 Student fellow Division of Molecular Hematology, A.O. Ospedali Riuniti di Bergamo -

Italy

2000-2009 Fellow, Division of Molecular Hematology, A.O. Papa Giovanni XXIII Bergamo - Italy

Technical skills and competences:

Molecular Biology: RNA and DNA preparation, RT-PCR, PCR and real time PCR, DNA sequence.

specialist of molecular diagnosis of myeloid and lymphoid Leukemias and molecular monitoring of minimal

Publications

1. Bassan R, Rossi G, Pogliani EM, Di Bona E, Angelucci E, Cavattoni I, Lambertenghi-Deliliers

G, Mannelli F, Levis A, Ciceri F, Mattei D, Borlenghi E, Terruzzi E, Borghero C, Romani C,

Spinelli O, Tosi M, Oldani E, Intermesoli T, Rambaldi A.

Chemotherapy-phased imatinib pulses improve long-term outcome of adult patients with

Philadelphia chromosome-positive acute lymphoblastic leukemia: Northern Italy Leukemia

Group protocol 09/00.

J Clin Oncol. 2010 Aug 1;28(22):3644-52. 20606084.

2. Bassan R, Spinelli O, Oldani E, Intermesoli T, Tosi M, Peruta B, Rossi G, Borlenghi E, Pogliani

EM, Terruzzi E, Fabris P, Cassibba V, Lambertenghi-Deliliers G, Cortelezzi A, Bosi A,

Gianfaldoni G, Ciceri F, Bernardi M, Gallamini A, Mattei D, Di Bona E, Romani C, Scattolin

AM, Barbui T, Rambaldi A.

Improved risk classification for risk-specific therapy based on the molecular study of

minimal residual disease (MRD) in adult acute lymphoblastic leukemia(ALL).

Blood. 2009 Apr 30;113(18):4153-62.

3. Spinelli O, Peruta B, Tosi M, Guerini V, Salvi A, Zanotti MC, Oldani E, Grassi A, Intermesoli T,

Micò C, Rossi G, Fabris P, Lambertenghi-Deliliers G, Angelucci E, Barbui T, Bassan R,

Rambaldi A.

Clearance of minimal residual disease after allogeneic stem cell transplantation and the

prediction of the clinical outcome of adult patients with high-risk acute lymphoblastic

leukemia.

Haematologica.2007 May;92(5):612-8. PubMed PMID: 17488684.

Addendum A - PERSONNEL INVOLVED IN THE RESEARCH - CURRICULUM VITAERambaldi Alessandro [PI]


Addendum B - BUDGET FORM AND JUSTIFICATIONSRambaldi Alessandro [PI]


Manuscript no. HAEMATOL/2010/031070 entitled “Inflammation and thrombosis inessential thrombocythemia and polycythemia vera: different role of C-reactiveprotein and pentraxin 3 “

Authors: Tiziano Barbui, Alessandra Carobbio, Guido Finazzi, Alessandro M.Vannucchi, Giovanni Barosi, Elisabetta Antonioli, Paola Guglielmelli, AlessandroPancrazi, Silvia Salmoiraghi, Pio Zilio, Cosimo Ottomano, Roberto Marchioli, IvanCuccovillo, Barbara Bottazzi, Alberto Mantovani, and Alessandro Rambaldi

Information about the contributions of each person named as having participated inthe study

1) Guarantor(s), i.e., person(s) who is (are) responsible for the integrity of the work as a whole:• Tiziano Barbui

According to the International Committee of Medical Journal Editors (ICMJE)(http://www.icmje.org/ethical_1author.html): “Authorship credit should be based on: 1)substantial contributions to conception and design, acquisition of data, or analysis andinterpretation of data; 2) drafting the article or revising it critically for important intellectualcontent; and 3) final approval of the version to be published. Authors should meet conditions 1,2, and 3 ……………………. Acquisition of funding, collection of data, or general supervision of theresearch group alone does not constitute authorship”.

The guarantors of this manuscript confirm that all persons designated as authors qualify forauthorship, and that each author has participated sufficiently in the work to take publicresponsibility for appropriate portions of the content.

2) Authors who participated in the conception of the study: Tiziano Barbui, Alessandro M.Vannucchi, Giovanni Barosi, Alberto Mantovani, and Alessandro Rambaldi

3) Design & Methods. The following authors were responsible for specific investigations (pleasedetail):• Alessandra Carobbio was responsible for statistical methods• Elisabetta Antonioli, Paola Guglielmelli, Alessandro Pancrazzi were responsible for collectingclinical data• Silvia Salmoiraghi, Pio Zilio, Cosimo Ottomano, Ivan Cuccovillo and Barbara Bottazzi wereresponsible for collecting laboratory data

4) Results. The following authors were responsible for specific portions of the results, includingfigures and tables (please indicate the person responsible for each figure and each table):• Alessandra Carobbio, Guido Finazzi, Roberto Marchioli and Tiziano Barbui were responsible forTable 1• Alessandra Carobbio, Guido Finazzi, Roberto Marchioli and Tiziano Barbui were responsible forTable 2• Alessandra Carobbio, Guido Finazzi, Roberto Marchioli and Tiziano Barbui were responsible forTable 3• Alessandra Carobbio, Guido Finazzi, Roberto Marchioli and Tiziano Barbui were responsible forTable and Figure in the On-line Appendix

5) Writing the manuscript. The following authors were responsible for writing the manuscript:• Tiziano Barbui, Alessandra Carobbio, Guido Finazzi, Alessandro M. Vannucchi, Giovanni Barosi,Roberto Marchioli, Ivan Cuccovillo, Barbara Bottazzi, Alberto Mantovani, and AlessandroRambaldi were responsible for revising the drafts and writing the final version of the paper

6) Contributors Listed in Acknowledgments: NA

Addendum C - PUBLICATIONS OF THE PIRambaldi Alessandro [PI]


Addendum D - BIO-ETHICAL REQUIREMENTS: Research on humans (Clearance from Ethics Committee)Rambaldi Alessandro [PI]


USC EMATOLOGIA

Direttore Prof. Alessandro Rambaldi

NOTA INFORMATIVA PER LA RACCOLTA, LA CONSERVAZIONE E L’USO

DI MATERIALE BIOLOGICO

Gentile Signore/Signora,

oggi la ricerca medica rappresenta una parte integrante delle attività di diagnosi e cura delle malattie e

compie costantemente grandi passi avanti, soprattutto grazie alle nuove tecnologie che permettono di

analizzare nei dettagli molecolari i processi biologici che le determinano. La possibilità di decodificare il

DNA (il codice della vita) ha permesso di individuare molti di tali dettagli, ma ancora molto deve essere

fatto per comprendere appieno e quindi curare le malattie.

Per poter eseguire tali ricerche è indispensabile avere a disposizione i materiali biologici dei pazienti

affetti da queste malattie; solo così sarà possibile capire quali sono i meccanismi molecolari e genetici che

determinano la loro insorgenza. Una volta identificate le alterazioni molecolari fondamentali che causano

i tumori, sarà possibile studiare nuove molecole in grado di interferire con tali alterazioni molecolari per

ripristinare le condizioni di normalità. In gergo scientifico si parla di terapie mirate (o terapia a bersaglio)

per sottolineare come i nuovi farmaci sono studiati per interferire con uno specifico “bersaglio

molecolare”; tali farmaci oltre ad essere più efficaci rispetto alle terapie convenzionali, sono più selettivi

e quindi producono minori effetti collaterali rispetto ai farmaci chemioterapici oggi utilizzati.

Attualmente i campioni biologici che Le vengono prelevati sono analizzati ai fini diagnostici nei

Laboratori dell’Unità di Ematologia: qui vengono sottoposti ad analisi al microscopio per classificare la

malattia sulla base del loro aspetto (diagnosi istologica) e sulla base delle loro caratteristiche molecolari

(diagnosi immunoistochimica, biomolecolare e citofluorimetrica). Il resto dei campioni non utilizzato ai

fini diagnostici, viene smaltito e distrutto come rifiuto speciale. Tale materiale in eccedenza può tuttavia

rappresentare una miniera di informazioni per la ricerca medico-scientifica.

Per questo motivo l’Unità di Ematologia si propone di raccogliere e conservare questi materiali in

eccedenza, per poterli utilizzare a scopo di ricerca medica presso i due Laboratori Divisionali: il

Laboratorio di Terapia Celulare “G. Lanzani” e il Laboratorio di Diagnostica Ematologica.

Per preservarne l’integrità i campioni vengono conservati a temperature estremamente basse. A tal scopo i

Laboratori dispongono di alcuni particolari congelatori nei quali i materiali biologici sono conservati a

temperature variabili da – 80° C (freezer meccanici) a – 196° C (contenitori con azoto liquido). Inoltre

dispongono di un sistema informatico che gestisce tutti i dati relativi ai materiali conservati.

Se Lei acconsente il materiale biologico rimanente al termine delle normali analisi di routine che Lei

effettuerà potrà essere conservato e analizzato a scopo di ricerca scientifica per un tempo non superiore a

20 anni. Nel caso Lei non acconsenta il materiale eccedente verrà smaltito e distrutto.

Per tutelare la riservatezza Le garantiamo che tutti i campioni raccolti saranno trattati in forma anonima e

nel rispetto delle condizioni descritte qui sotto.

Nel caso Lei acconsenta, il materiale biologico sarà soggetto alle seguenti condizioni di utilizzo:

il campione raccolto rimanente sarà conservato presso il Laboratorio di Terapia Celulare “G. Lanzani”

e/o presso il Laboratorio di Diagnostica Ematologica con le procedure idonee a garantirne l’integrità;

AZIENDA OSPEDALIERA

OSPEDALI RIUNITI DI BERGAMO

di rilievo nazionale e di alta specializzazione



il materiale biologico ed i relativi dati potranno essere utilizzati e/o ceduti (in forma anonima) a

Istituti universitari o ad altri Istituti di ricerca italiani o esteri per il solo fine di ricerca scientifica e

solo nell’ambito di ricerche rispettose della dignità umana, previa approvazione del Comitato Etico

competente;

il campione ed i relativi dati potranno essere utilizzati ad esclusivo scopo di ricerca scientifica e mai a

scopo di lucro diretto. Non potranno dunque essere oggetto di compravendita, come espressamente

vietato dalla Convenzione di Oviedo del 1997. Tuttavia nei limiti e nelle forme previste dalla legge 22

febbraio 2006, n. 78 (riguardante i limiti e condizioni per la brevettabilità del corpo umano) una

invenzione ottenuta a partire dai campioni biologici conservati presso i due Laboratori potrebbe essere

oggetto di brevetto, il cui sfruttamento potrebbe originare dei profitti per l’Ente che ha effettuato la

scoperta. La concessione del brevetto anche in ambito di ricerca medica è un riconoscimento

all’attività dell’inventore; gli eventuali profitti derivanti dallo sfruttamento del brevetto sono pertanto

considerati il frutto dalla capacità tecnico-scientifica di chi ha svolto la ricerca e quindi saranno gestiti

e apparterranno unicamente a tale Istituzione;

i dati derivanti dai campioni saranno trattati nel rispetto del D.Lgs. 196/2003 nonché

dall’Autorizzazione al trattamento dei dati genetici emanata dal Garante per la protezione dei dati

personali. In particolare i dati ed i relativi campioni saranno trattati esclusivamente da personale

autorizzato dai Responsabili dei Laboratori e l’accesso ai sistemi informatici e ai locali ove essi sono

custoditi sarà controllato mediante idonee misure di sicurezza. Saranno adottate tutte le misure

tecnologiche idonee a prevenire la diffusione dei dati personali o il loro utilizzo da parte di soggetti

non autorizzati;

i campioni ed i dati ad essi relativi vengono gestiti in forma codificata, ossia mediante l’attribuzione

di un codice assegnato da un sistema informatico. I ricercatori che studieranno i campioni avranno a

disposizione campioni e dati contraddistinti unicamente dal codice segreto, che impedisce loro

qualsiasi possibilità di associare i dati delle indagini scientifiche con l’identità dei donatori. Il

Responsabile del laboratorio, o un suo delegato, potranno attivare una procedura per associare i dati e

i campioni alla identità dei donatori solo in due situazioni: quando sia indispensabile per condurre uno

specifico progetto di ricerca o quando risponda a precise esigenze cliniche nell’interesse del donatore;

i Laboratori non si ritengono responsabili per eventuali danni o incidenti che possano verificarsi sui

campioni conservati e si riservano la facoltà di eliminare in qualsiasi momento il campione ed i dati

ad esso relativi;

la diffusione dei dati scientifici potrà avvenire solo in forma anonima per sole finalità scientifiche. In

pratica, i risultati delle ricerche scientifiche, potranno essere presentati nell’ambito di Convegni

ovvero su riviste specializzate senza mai permettere la precisa identificazione dei pazienti donatori.

Spesso peraltro i dati scientifici sono il risultato della analisi di grandi gruppi di campioni biologici,

provenienti da numerosissimi pazienti, per cui i risultati finali potranno essere presentati e pubblicati

in forma aggregata. Ad esempio, la identificazione di una mutazione genetica significativa per un dato

tipo di tumore può essere ottenuta solo analizzando centinaia di campioni diversi dello stesso tipo

tumorale. La divulgazione di tale scoperta sarà pertanto effettuata descrivendo la sua frequenza

all’interno di un dato gruppo di pazienti e la relazione tra mutazione, evoluzione clinica e risposta alle

terapie;

in caso in cui l’attività di raccolta e bancaggio campioni dovesse essere interrotta, la corretta

conservazione e utilizzo dei campioni sarà garantita dal Direttore dell’Unità di Ematologia degli

Ospedali Riuniti di Bergamo a cui afferiscono i Laboratori;

in ogni momento Lei potrà comunicare eventuali cambiamenti di opinione in merito a quanto

dichiarato. Nel caso desideri accedere alle informazioni relative ai propri campioni biologici, o

desideri ottenere ulteriori informazioni, si potrà rivolgere ai Responsabili dei Laboratori: Dott.

Martino Introna (Laboratorio di Terapia Cellulare G. Lanzani) e Dott.ssa Orietta Spinelli (Laboratorio

di Diagnostica ematologica) o comunque al Direttore dell’Unità di Ematologia Prof. Alessandro

Rambaldi presso l’Azienda Ospedaliera Ospedali Riuniti di Bergamo. Tutte le informazioni raccolte

saranno trattate nel rispetto della normativa italiana sulla tutela dei dati personali (D.Lgs. 196/2003)

ed il trattamento dei suoi dati è avviato con la sottoscrizione del consenso informato ed al trattamento



dei dati personali, previa lettura della presente Nota informativa. Ogni partecipante ha, in ogni

momento, facoltà di esercitare i diritti di cui all’art. 7 del D.Lgs. 196/2003, qui di seguito riportato:

Diritto di accesso ai dati personali ed altri diritti

1. L'interessato ha diritto di ottenere la conferma dell'esistenza o meno di dati personali che lo

riguardano, anche se non ancora registrati, e la loro comunicazione in forma intelligibile.

2. L'interessato ha diritto di ottenere l'indicazione:

a) dell'origine dei dati personali;

b) delle finalita' e modalita' del trattamento;

c) della logica applicata in caso di trattamento effettuato con l'ausilio di strumenti elettronici;

d) degli estremi identificativi del titolare, dei responsabili e del rappresentante designato ai sensi

dell'articolo 5, comma 2;

e) dei soggetti o delle categorie di soggetti ai quali i dati personali possono essere comunicati o che

possono venirne a conoscenza in qualita' di rappresentante designato nel territorio dello Stato, di

responsabili o incaricati.

3. L'interessato ha diritto di ottenere:

a) l'aggiornamento, la rettificazione ovvero, quando vi ha interesse, l'integrazione dei dati;

b) la cancellazione, la trasformazione in forma anonima o il blocco dei dati trattati in violazione di

legge, compresi quelli di cui non e' necessaria la conservazione in relazione agli scopi per i quali i

dati sono stati raccolti o successivamente trattati;

c) l'attestazione che le operazioni di cui alle lettere a) e b) sono state portate a conoscenza, anche per

quanto riguarda il loro contenuto, di coloro ai quali i dati sono stati comunicati o diffusi,

eccettuato il caso in cui tale adempimento si rivela impossibile o comporta un impiego di mezzi

manifestamente sproporzionato rispetto al diritto tutelato.

4. L'interessato ha diritto di opporsi, in tutto o in parte:

a) per motivi legittimi al trattamento dei dati personali che lo riguardano, ancorche' pertinenti allo

scopo della raccolta;

b) al trattamento di dati personali che lo riguardano a fini di invio di materiale pubblicitario o di

vendita diretta o per il compimento di ricerche di mercato o di comunicazione commerciale.

Il Titolare del Trattamento dei dati personali (ex. D.lgs 196/2003) è il Dott. Carlo Nicora

Il Responsabile del Trattamento dei dati personali (ex. D.lgs 196/2003) è il Prof. Alessandro Rambaldi

In conclusione, ci auguriamo che questo documento redatto in accordo alle “Linee Guida” della

Presidenza del Consiglio dei Ministri, messo a punto dal Gruppo congiunto (Comitato Nazionale di

Bioetica e Comitato Nazionale per le Biotecnologie e Scienze della Vita) ) e in accordo con i

suggerimenti del Centro Nazionale di Epidemiologia, Sorveglianza e Protezione della Salute dell’ Istituto

Superiore di Sanità, possa aver con chiarezza illustrato i motivi che ci spingono a conservare e studiare il

materiale biologico ottenuto dai nostri pazienti.

Non esiti a contattarci per qualunque aspetto richieda ulteriori chiarimenti da parte nostra ai recapiti sotto

riportati.

Grazie per la collaborazione

Unità di Ematologia


A. O. Ospedali Riuniti di Bergamo, L.go Barozzi, 1 – 24128, Bergamo tel. 035.269492 fax 035.266147

Laboratorio di Terapia Cellulare “G. Lanzani”

Responsabile Dott. Martino Introna

Presidio Matteo Rota, Via G. Garibaldi, 11/13 – 24124, Bergamo tel. 035.2278684 fax 035.2278674

Laboratorio di Diagnostica Ematologica

Dott.ssa Orietta Spinelli

A. O. Ospedali Riuniti di Bergamo, L.go Barozzi, 1 – 24128, Bergamo tel. 035.266810 fax 035.266659



USC EMATOLOGIA


MODULO PER L’ACQUISIZIONE DEL CONSENSO INFORMATO

PER LA RACCOLTA, LA CONSERVAZIONE E L’USO DI MATERIALE BIOLOGICO

Il sottoscritto/a ________________________________ nato/a a _________________ il __/__/____

Residente in _____________________________________ CAP _____________ Prov. _________

Via _____________________________________ n° _______ tel.___________________________

dichiara di:

aver letto e compreso tutte le informazioni, anche per la parte relativa al “Trattamento dei dati personali”, riportate nella Nota Informativa relativa a: “raccolta, conservazione e uso di materiale biologico”, di cui ha ricevuto una copia da conservare.

essere consapevole che la propria partecipazione è libera, gratuita e volontaria e che ci si può spontaneamente ritirare, avendo ricevuto la certezza che nè il rifiuto alla donazione, nè l’eventuale ritiro della adesione comporteranno discriminazioni.

aver ricevuto garanzia che per ulteriori informazioni potrà rivolgersi al Responsabile del trattamento dati personali* per la conservazione dei campioni biologici o persona da lui designata.

essere stato informato che il materiale biologico prelevato nel corso dello studio ...............................………......, potrà essere conservato presso il Laboratorio di Terapia Cellulare “G. Lanzani”, e/o presso il Laboratorio di Diagnostica Ematologica afferenti all’Unità di Ematologia per un tempo non superiore a 20 anni, sotto la diretta responsabilità dei Responsabili dei Laboratori (Dott. Martino Introna del Laboratorio di Terapia Cellulare “G. Lanzani”, e dott.ssa Orietta Spinelli del Laboratorio di Diagnostica Ematologica).

essere stato informato che il materiale biologico prelevato potrà essere utilizzato per ulteriori indagini (oltre a quelle previste nell’ambito dello studio .................…….....................), comunque sempre a scopo esclusivo di ricerca in campo onco-ematologico finalizzato al miglioramento delle procedure diagnostiche, prognostiche, predittive e terapeutiche, mai a fini di lucro diretto, e che le ricerche suddette saranno previamente sottoposte al vaglio del Comitato Etico competente.

essere stato informato che saranno attuate tutte le misure atte a garantire la riservatezza e la anonimizzazione reversibile (codifica e criptazione) del materiale biologico, sia nell’ambito delle indagini sia nella raccolta e conservazione dei campioni, in base alla vigente normativa.

essere stato informato che saranno attuate tutte le procedure idonee a garantire l’idoneità del materiale biologico conservato; nonostante ciò, il Laboratorio di Terapia Cellulare “G. Lanzani” e il Laboratorio di Diagnostica Ematologica non si ritengono responsabili per eventuali danni e incidenti che possano verificarsi e esitare nella perdita del materiale biologico conservato.

aver ricevuto garanzia che in ogni momento potrà comunicare eventuali cambiamenti di opinione in merito a quanto autorizzato; e che in caso di ritiro dell’adesione, il campione ed i relativi dati saranno eliminati e non potranno essere utilizzati per future ricerche, oppure verranno (su richiesta dell’interessato) resi irreversibilmente anonimi per cui non sarà più possibile identificare il donatore.

AZIENDA OSPEDALIERA

OSPEDALI RIUNITI DI BERGAMO

di rilievo nazionale e di alta specializzazione



Pertanto:

1 autorizza / non autorizza

la raccolta, conservazione e l’uso del materiale biologico qui sotto specificato, secondo le modalità

previste dall’Informativa

sangue periferico sangue midollare altro_________________________________

appartenente a:

se stesso

_______________________________ di cui il sottoscritto/a è _________________________*

nome e cognome genitore/tutore

* Al momento del compimento dei 18 anni, questo consenso verrà risottomesso all’attenzione e alla firma

del diretto interessato

2 acconsente / non acconsente

all’utilizzo di detto materiale biologico per ricerca in campo onco-ematologico come sopra specificato per

studi promossi e/o condotti dall’Unità di Ematologia previamente approvati dal Comitato Etico competente


alla comunicazione, nei Suoi confronti, di eventuali risultati che abbiano valore clinico e/o diagnostico

derivanti dai suddetti studi e ricerche


al trattamento dei dati personali, anche di carattere genetico relativi al materiale in oggetto, per le finalità

di raccolta, mantenimento e utilizzo del materiale biologico (in base alle disposizioni del d.lgs. 196/2003)


all’eventuale cessione (in forma anonima) dei dati personali e/o del campione biologico a Istituti

universitari o ad altri Istituti di ricerca italiani o esteri per il solo fine di ricerca scientifica;

Bergamo, li |__|__| / |__|__| / |__|__|__|__| Firma dell’interessato/a ___________________________

Per l’ interessato/a se non è in grado di firmare

Bergamo, li |__|__| / |__|__| / |__|__|__|__| Firma __________________________________________ genitore o tutore

Bergamo, li |__|__| / |__|__| / |__|__|__|__| Firma __________________________________________ genitore o tutore

Il Sanitario che ha raccolto il consenso: cognome e nome _____________________________________

Bergamo, li |__|__| / |__|__| / |__|__|__|__| Firma__________________________________________ firma e timbro

I Responsabili dei Laboratori:

Dott. Martino Introna per il Laboratorio di Terapia Cellulare “G. Lanzani”,

Dott.ssa Orietta Spinelli per il Laboratorio di Diagnostica Ematologica

garantiscono il rispetto delle suddette dichiarazioni.



CERTIFICATO DI PUBBLICAZIONE

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Pubblicata all’Albo Pretorio on-linedell’Azienda Ospedaliera

“Papa Giovanni XXIII” Bergamo

per 15 giorni

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DELIBERAZIONE N. 602/2015 ADOTTATA IN DATA 16/04/2015 · 2015-04-20 · The new HTS based method...

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