TP53 ABERRATIONS Methodical considerations · 2019-12-12 · TP53 aberrations: Methodical considerations TP53 gene in CLL: KEEP IN MIND In CLL, TP53 mutations are (1) frequently subclonal,

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 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


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


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



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


HOW: which gene parts should be covered

OPTIMUM: WHOLE CODING REGION = EXONS 2-11

Leroy etal., Human Mutation 2014; Pospisilova and Pettitt, 2010



Leroy etal., Human Mutation 2014; Pospisilova and Pettitt, 2010 Thierry Soussi, The TP53 Web Page

OPTIMUM: WHOLE CODING REGION = EXONS 2-11



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 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


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


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


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 web site/Seshat tool http://p53.fr


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


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


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

http://varnomen.hgvs.org/

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


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

TP53 ABERRATIONS Methodical considerations · 2019-12-12 · TP53 aberrations: Methodical considerations TP53 gene in CLL: KEEP IN MIND In CLL, TP53 mutations are (1) frequently subclonal,

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