Significato clinico e applicazioni dell’analisi dei minor

BCR-ABL1 breakpoints

Orietta Spinelli

Laboratorio di Ematologia “Paolo Belli”

USC Ematologia

Ospedale Papa Giovanni XXIII

Bergamo

Milano, 13 Novembre 2014

m-bcr

p190

M-bcr

p210

Usefulness of BCR-ABL identification

Diagnostic

Prognostic Therapeutic

pre and post TKI

CML within MPNs

Ph+ ALL within ALL

Imatinib: How it works?

TKI + chemotherapy + SCT

TKI + chemotherapy

At CR

At 6 month

At 12 month

Technique SensitivityDisease

PhaseAdvantages Disadvantages

Conventional

caryotype10-2 Diagnosis

-accompaining

alterations

-low sensitive

- criptic rearrangements

FISH 10-2 Diagnosis -cryptic rearrangements -low sensitive

Qualitative PCR10-3

(2nd 10-5)

Diagnosis

(follow-up)

-cryptic rearrangements-2 amplification rounds for

high sensitivity

-rare transcripts

-prone to test

contaminations (2nd

round-nested PCR)

-relatively cheap

-prone to lab

contaminations (post

amplification

manipulations)

Quantitative

PCR10-5 (Diagnosis)

follow-up

-high sensitive w 1

round-no rare transcripts

-no manipulation post

amplifications-higher costs (plasmids)

- quantification - quantification

Crucial points

Sample type (BM vs PB)

WBC vs mononuclear cells (buffy+ lysis vs ficoll)

Sample Quality volume (BM and PB)

time to processing

RNA quality extraction method

260/280 ratio

RIN

PCR design aspecific amplification

contaminations

CG

RQ-PCR design aspecific amplification ( less frequent w probe)

CG espression level and stability (in different tissues

and treatment phases)

plasmid contamionations

CG interference

EAC cDNA and Qualitative PCR

*

* Superscript II

EAC PCR for p190

EAC PCR for p210

P190 PCR amplification P210 PCR amplification

Quantitative PCR

EAC

RT and RQ-PCR

protocols

EAC RQ-PCR

p190 and p210

Standard curve and copy number calculation

Plasmid diluition at known

copy number content

(from 106 to 10 copies)

Copy number calculation

based on sample Cycle

threshold (Ct) value

10e6 10e5 10e4 10e3 10e2 10

Sample Ct =27,5

5 x 104

102 103 104 105 106

IPSOGEN FGRS-9 (p190) /FGRS-10 (p210)

10e6 10e5 10e3 10e2 10

IPSOGEN CGRS-1 (ABL)

10e5 10e4 10e3

ABLBCR-ABL

Transcript quantification

Sample: BCR-ABL and ABL levels

1- BCR-ABL Normalized Copy Number (NCN BCR-ABL)

NCN BCR-ABL = Target Copy Number x 104

CG Copy NumberAt least 10000 ABL copies

2- BCR-ABL %

BCR-ABL % = Target Copy Number x 100

CG Copy Number

3- BCR-ABL International Scale %

BCR-ABL IS % = Target Copy Number x 100 x CF*

CG Copy Number

*CF Convertion Factor experimentally calculated for Major BCR-ABL1 in

CML based on reference sample/material

Transcript quantification

International Quality Controls:

EUTOS

for Major BCR-ABL1 in CML

LabNet:

CF calculation for italian partecipating laboratories for Major BCR-ABL1 in CML

Referece laboratories:

UO di Ematologia, Università Federico II di Napoli & CEINGE

Biotecnologie Avanzate

Reference Person: Prof Fabrizio Pane

Dipartimento di matologia e Oncologia “L. & A: Seragnoli dell’Università

di Bologna

Reference Persons: Prof. Michele Baccarani

Prof. Giovanni Martinelli

Dott. Gianantonio Rosti

Dipartimento di Scienze Cliniche e Biologicheliniche e Oncologia

dell’Università di Torino

Reference Person: Prof. Giusepe Saglio

Partecipating laboratories: 31 laboratories in 2011

minor BCR-ABL1?

EuroMRD: European Study Group

on MRD detection

AIMS of EuroMRD (focus on PCR ananlysis of Ig/TCR genes):

1. Quality Control Program: 2 times per year: - February / March

- August / September

2. Educational meetings, including evaluation of quality control rounds:

2 times per year: - May / June

- October / November

3. Standardization of MRD techniques

- standardization techniques within each treatment protocol

- guidelines for interpretation of RQ-PCR results

- standardized diagnostics reports

4. Collaborative development and clinical evaluation of new MRD

strategies and new MRD techniquesBasis for accreditation of laboratory diagnostics

and minor-BCR-ABL (Coordinator H. Pfifer, Frankfurt)

CONCLUSIONS

- Identification of t( 9;22) (q34;q11) and BCR-ABL transcripts is

mandatory for diagnostic, prognostic and therapeutic issues in

CML and ALL.

- Qualitative PCR for BCR-ABL identification is preferable at

diagnosis to identify possible transcript variants

- Quantitative PCR is the gold standard for MRD evaluation

- Potential pitfalls in BCR-ABL evaluation are sample quality,

RNA degradation and contaminations

- Quality controls are recommended to ensure accurate and

reliable results