TP53 ABERRATIONS Methodical considerations
Sarka Pavlova
University Hospital and Masaryk University, Brno, Czech republic
Belgrade March 16-17, 2018
N=228
mutation & 17p deletion
FISH pos
17p deletion without mutation
mutation(s) without 17p deletion
FISH neg
TP53 aberrations: Methodical considerations
TP53 gene in CLL: modes of inactivation
mutation/cn-LOH
monoallelic mutation
N=52 (SNP array)
Examine not only del(17p) by
FISH but also TP53 mutation
Karla Plevova
TP53 aberrations: Methodical considerations
TP53 gene in CLL: KEEP IN MIND
In CLL, TP53 mutations are (1) frequently subclonal, (2) clinically significant even if the second allele remains intact KNOW DETECTION LIMIT OF YOUR METHOD AND CANCER CELL FRACTION
Landau et al., Cell 2013
Mutations can NEWLY OCCUR IN RELAPSE
Pre-therapy Relapse 1 Pre-therapy Relapse 1 Relapse 2-n
SangerSeq detection limit
Malcikova et al., Leukemia 2015
Despite some codons are mutated more frequently (hotspots), deleterious variants can occur IN ANY PART OF CODING SEQUENCE
Leroy etal., Human Mutation 2014 Pospisilova and Pettitt, 2010
TP53 aberrations: Methodical considerations
Part I From clinician to lab: SAMPLING
Part III From lab to clinician: REPORTING
WHEN to sample? WHAT type of tissue? INFORMATION which should be provided
ALWAYS KEEP GENERAL STANDARDS OF LABORATORY PRACTICE: strictly avoid sample swap/cross-contamination at any phase and include appropriate controls
clear and precise message from lab to clinician
Gene analysis: technical view
Keep communicating
Part II In laboratory
WHICH CELLS to enrich for? DNA or RNA? WHICH GENE PARTS to analyze? METHOD? SANGER SEQ: protocol and analysis? NGS: what to consider? CORRECT VARIANT DESCRIPTION INTERPRETATION: does the variant affect function?
PERIPHERAL BLOOD
TP53 aberrations: Methodical considerations
Part I From clinician to lab
2. WHAT type of tissue?
Optionally - if cancer cell fraction (CCF) is low in PB (SLL/CLL) • BONE MARROW • LYMPH NODE: fresh (frozen) FFPE not suitable unless specific methodology is established
IF DECIDING ABOUT TREATMENT
1. WHEN to analyze TP53 during disease course?
= before first therapy and before every subsequent therapy
Not necessary – if the information does not impact patient’s management: • mutation documented previously • p53-independent therapy cannot be given (comorbidities, limited access) ? if targeted therapy is given irrespective of TP53 status
TP53 aberrations: Methodical considerations
Part II In laboratory
WHICH CELLS to enrich for?
MONONUCLEAR FRACTION works for majority of samples
Optionally for <60-70% lymphocytes: • CD19+ separation is recommended
e.g. RosetteSep or MACS, magnetic-activated cell sorting or FACS
• use sensitive method (deep NGS) and recalculate VAF
density gradient centrifugation using appropriate media, i.e. Ficoll or Histopaque 1077
CLL cells = Cancer cell fraction (CCF)
CLL: WBC 14.7x109/L lymphocytes 59%
CD 5
CD
19
1 %
normal B-cells
CLL 35%
43 % CD5+ T-cells
21 % 5-19- MNC cells
MONONUCLEAR CELLS (gradient centrifugation)
CD 5
CD
19
2 % normal B-cells
CLL >95%
<1 % CD5+ T-cells
1 % 5-19- MNC cells
B-CELLS (non B-cell
depletion by RosetteSep)
Cancer Cell Fraction (CCF):
CLL = 20%
LEUKOCYTES
p.Y220C gDNA 8% p.Y220C gDNA 21%
CONSIDER CANCER CELL FRACTION either during sample processing or result interpretation (esp. CLL/SLL)
INFORMATION ON BLOOD CELL COUNT parameters (at least WBC) provided with the sample may be helpful
WHICH CELLS to enrich for? Cancer cell fraction (CCF)
Mutation VAF
DNA of standard quality
TP53 aberrations: Methodical considerations Part II In laboratory
NUCLEID ACID isolation and storage
• DNA storage (deep NGS): in TE buffer with low EDTA (0.1mM) at -20°C for longer periods avoid unnecessary dilution • For NGS, low concentration or poor quality low amount of template
molecules in the sample false negative and false positive results
TP53 aberrations: Methodical considerations Part II In laboratory
HOW: which gene parts should be covered
OPTIMUM: WHOLE CODING REGION = EXONS 2-11
Leroy etal., Human Mutation 2014; Pospisilova and Pettitt, 2010
TP53 aberrations: Methodical considerations Part II In laboratory
HOW: which gene parts should be covered
Leroy etal., Human Mutation 2014; Pospisilova and Pettitt, 2010 Thierry Soussi, The TP53 Web Page
OPTIMUM: WHOLE CODING REGION = EXONS 2-11
TP53 aberrations: Methodical considerations Part II In laboratory
HOW: which gene parts should be covered
minimum: exons 4-10
Thierry Soussi, The TP53 Web Page
ALWAYS INCLUDE SPLICE SITES intronic +/- 2 bp from intron/exon boundaries
Splice site variants are pathogenic
Part II In laboratory Sanger seq only
38%
NGS only 25%
Sanger sequencing and
NGS 28%
Sanger seq, NGS and
other 6%
I am not sure 1%
Other only 2%
METHOD OF ANALYSIS
TP53 Sanger sequencing
Next generation Sequencing - NGS
Easy and cheap to
establish for any lab using capillary sequencing
Running few samples per month
Fast
Subclonal variants with 10-20% VAF may escape
Incorrect quantification for some variants
Easily detects all 10-20% mutations if properly
established Quantitative High-throughput method – large cohorts More genes/diseases at once in a gene panel Research on clonal evolution – minor
mutations <<10% can be caught Takes longer High-throughput = expensive or slow for low
sample numbers or if not combined with other genes/diseases
If low-VAF variants desired, higher coverage must be applied for all genes in the panel
ERIC/Gilead survey
01/2014 Before therapy
06/2016 Progression
11/2015 Complete remission
R-Bendamustine 4x
del(17p) neg p.G105D 2.5% VAF
cell separation CLL 15%96% TP53 mutations 70% VAF
p.G105D 61% VAF + 6 other mutations 2.9%-0.5%
del(17p) neg, cn-LOH 80% TP53 mutations 76% VAF
p.G105D 68% VAF + 6 other mutations 2.1%-0.5%
FCR 4x efekt PR
02/2014 Before therapy
11/2016 Progression
del(17p) neg c.128delT 1.5%
Ibrutinib
NGS IN RESEARCH ON CLONAL EVOLUTION
del(17p) neg c.128delT 4.2% p.R248Q 1.5%
?
Minor clones may evolve into
prevalent refractory clone under therapy pressure (but not always do)
Landau et al., 2013; Rossi et al., 2014; Malcikova et al. 2015….
Convergent mutated subclones occur in parallel Jehtwa et al., 2013; Malcikova et al. 2015..
Jitka Malcikova, Jana Kotaskova
TP53 aberrations: Methodical considerations Part II In laboratory
TP53 Next Generation Sequencing
Standalone assay or gene panel Amplicon-based or capture-based Unique molecular identifiers (UMI) may be helpful Minimum limit of detection Sanger seq, i.e. 10% or less Corresponding coverage at all analyzed positions (to reach at least 10 reads per
variant) and DNA/allelic input (6 pg per human cell)
FALSE NEGATIVE RESULTS
• Low/non-uniform coverage • Low DNA input or quality –
sampling effect • Inefficient capture low DNA
input • Variant excluded/not recognized
by bioinformatics pipeline (sub-optimal alignment or variant calling)
FALSE POSITIVE RESULTS • Background noise are higher
than your expectations • Sequencing errors • PCR errors • Non-proof reading polymerase • DNA from FFPE • False variant created or not
excluded during bioinformatics pipeline
library preparation
sequencing
bioinformatics pipeline
manual inspection and interpretation
TP53 aberrations: Methodical considerations
IARC p53 website protocol: • adaptable, can be modified • number of PCR cycles 5030 • some primers span rare
polymorphic sites • some primers very close to
exon/intron boundary
Part II In laboratory
Sanger sequencing of TP53 gene
SEQUENCING PROTOCOL: you may start at IARC p53 WEBSITE
ALWAYS SEQUENCE BOTH FORWARD AND REVERSE STRAND
http://p53.iarc.fr/ProtocolsAndTools.aspx
TP53 aberrations: Methodical considerations Part II In laboratory
Sanger sequencing of TP53 gene
Carefully go through chromatogram not to overlook subclonal variants
Free web-based software GLASS available via ERIC website
http://shiny.bat.infspire.org/glass/
Do not trust software completely and check the sequence manually
TP53 aberrations: Methodical considerations
TP53 web site/Seshat tool http://p53.fr
Part II In laboratory
Variant interpretation: does the variant affect function?
CHECK FUNCTIONAL IMPACT OF THE MUTATION IN GENE-SPECIFIC DATABASE (do not use dbSNP)
TP53 IARC database http:// p53.iarc.fr
IF YOU HAVE DOUBTS (RARE or FUNCTIONAL VARIANT), REPEAT THE ANALYSIS FROM PCR TO EXCLUDE ANALYTICAL ERRORS
SEVERAL WELL-DESCRIBED SNPs / NEUTRAL GERMLINE EXONIC VARIANTS OCCUR IN HUMAN POPULATION:
http://p53.iarc.fr/TP53GeneVariations.aspx
Part II In laboratory TP53 aberrations: Methodical considerations
Variant interpretation: does the variant affect function?
c.704A>G: p.Asn235Ser c.847C>T: p.Arg283Cys
our cases: 4/4 germline no personal or family history of Li-Fraumeni syndrome
NEVER REPORT THESE VARIANTS AS PATHOGENIC Ask for non-tumor DNA (buccal swab, T-cells etc.)
MOST OTHER VARIANTS DETECTED BY SANGER ARE DELETERIOUS BUT, VARIANTS THAT PROBABLY DOES NOT AFFECT FUNCTION
MAY RARELY OCCUR – OFTEN GERMLINE - RARE SNPs!
Part II In laboratory TP53 aberrations: Methodical considerations
Variant interpretation: does the variant affect function?
WT
RGC
p21
bax
R283C
RGC
p21
bax
MUT R175H
RGC
p21
bax
Adapted from Jagosova et al. Int J Oncol 2012
REPORT TEMPLATE – see ERIC webpage and ERIC Recommendations on TP53 Analysis
GIVE CLEAR RESULT - It is preferred not to include common polymorphisms in
the report to physicians.
REPORT VARIANTS DETECTABLE BY SANGER SEQUENCING AND VARIANTS PRESENT IN >10% VAF IF TESTED BY NGS “Reporting variants between 5-10% VAF is acceptable only if explicitly stating that the clinical impact of minor subclonal mutations has not been conclusively documented.”
! Low VAF in sample with low CCF – 5% VAF in sample with 20% CCF corresponds to 25% in sample with 100% purity
Reporting -clear and precise message from lab to clinician
TP53 aberrations: Methodical considerations Part III From lab to clinician: REPORTING
Use HGVS NOMENCLATURE:
http://varnomen.hgvs.org/
Report the cDNA and protein level including reference sequence (LRG_321 NM_00546)
NGS: pipelines do not often describe dup, ins and dels correctly, always inspect
NGS: notice if appropriate reference sequence is used
Reporting: variant description
TP53 aberrations: Methodical considerations Part III From lab to clinician: REPORTING
Do not change patient and sample IDs in various papers duplicities in databases
Include genomic coordinates and reference genome
List all variants, including synonymous and benign variants
Publishing and scientific reporting in databases
TP53 aberrations: Methodical considerations Final note: Publishing and databases
TP53 aberrations: Methodical considerations
THANK YOU FOR ATTENTION Jitka Malčíková Šárka Pospíšilová Michael Doubek Jana Kotašková Yvona Brychtová Lenka Juračková Anna Panovská Marcela Ženatová Jiří Mayer Lenka Kociánová clinical department Renata Hurníková staff Zuzana Bučková Karla Plevová Jana Šmardová Martin Trbušek Boris Tichý Nikola Tom Karol Pál cytogenetic and flowcytometry laboratory