MPN Diagnostics

Bone marrow histology features of MPN

That can be assessed with reasonable consistency:

Overall haemopoietic cellularity

Relative predominance of increased megakaryocyte numbers

Megakaryocyte distribution (not the cytological details)

Reticulin pattern

 

Fibrosis Grade

 

WHO 0-3

Bain 1-4

 

Grade of fibrosis predicts overall survival

PT-1 Trial – 5 yr OS – Approx 95% for grade 0/1, Approx 87% for grade 3/4

 

Phenotypic ‘Driver’ Mutations

mpn dx.png

 

CML Molecular genetics

 

Depending on the breakpoints in the BCR and ABL1 genes, different sized proteins will be formed from the fusion gene:

 

-       e1a2 protein - p190 (seen in ALL > CML)

-       b2a2/b3a2 proteins - p210 (most common in CML)

-       e19a2 protein - p230

 

This is important diagnostically, and need to know which transcript is present when comes to quantitating its presence by RT-PCR. Different transcripts will need different primers for the lab test à not all transcripts can be quantified in all labs.

 

JAK2 & MPL Mutations

 

Mutated JAK2/MPL is activated spontaneously without the presence of the usually required ligands à persistent activation of the downstream pathways – e.g. STAT, PI3K, MAPK

 

JAK2 V617F at CUH Lab

-       Quantitative PCR using a fluorescent probe that is released as the allele replicates

-       One probe for the mutant allele and one for the wild type

-       Compare fluorescence of each to quantify

 

MPL and JAK2 Exon 12 at CUH

-       High resolution melt profile performed after PCR has produced the protein

-       Difference in the way mutated protein melts at 84 degrees

 

CALR Mutation

 

Insertion or deletions in Exon 9 are the most common mutations.

CALR is an endoplasmic protein, mutation leads to abnormal movement of MPL within the cell

 

CALR at CUH Lab

-       Primer for the area of interest within the CALR gene

-       The length of the protein produced during replication will tell you if mutated – shorter or longer than the wild type (deletions or insertions)

 

NGS Panels

 

Next generation sequencing to test whole panels of genes in a single assay

Sensitive for detecting mutations at low levels

Can test several patients within one assay à reduces costs

 

Ion Torrent Analyser used at CUH

-       Currently for MF (not PV or ET)

 

Significance of results

-       Increasing no. of mutated genes detected in panel à predicts high molecular risk disease

 

Future of testing

-       BCR/ABL, JAK2, CALR, MPL may put into a single panel and run all at once

-       May not be quite a sensitive as individual JAK2 assay ?significance of this