INDICATIONS FOR GERMLINE TESTING FROM TUMOUR-ONLY … · Neuroendocrine Tumor 90 Retinoblastoma 12 Cancer of Unknown Primary 428 Cervical Cancer 87 Wilms Tumor 11 Ovarian Cancer 398

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INDICATIONS FOR GERMLINE TESTING FROM TUMOUR-ONLY SEQUENCING

Consensus Recommendations from ESMO Working Group

ACGS Birmingham 10th

June 2019

Clare Turnbull Professor in Genomic Medicine, Queen Mary University of London and Institute of Cancer Research

Clinical Lead for Cancer Genomics, 100,000 Genomes Project, Genomics England

Consultant in Cancer Genetics (Honorary), Guys and St Thomas and Barts NHS Trusts

Consultant in Public Health Medicine (Honorary), Public Health England

Clare Turnbull, Institute of Cancer Research, Queen Mary University London (Chair),

Diana Mandelker, Memorial Sloan Kettering

Angela George, Institute of Cancer Research/Royal Marsden

Funda Meric-Bersntam, MD Anderson

Emmanuele Rial-Sebbag, University of Tolouse

Susan Wallace, University of Leicester

Meetings: 6

Data analyses: Mandelker+team, Turnbull

WORKING GROUP

Early tumour testing:

Single mutation/hot-spots testing (genotyping)

Oncogenes

NGSrecent expansion in tumour testing:

Larger panels: more genes

More patients (umbrella/drug-matching trials)

Sequencing entire gene

Tumour suppressor genes (overlap with cancer susceptibility genes)

Approach

Tumour-normal paired sequencing (WGS, research)

Tumour-only (majority including commercial providers)

WHEN SHOULD A FINDING IN THE TUMOUR-ONLY SEQUENCNG

TRIGGER A GERMLINE TEST?

BACKGROUND Somatic==acquired==tumour only

Germline==constitutional

Association with tumour

On-tumour association: “pertinent” but mainly for aetiological reasons -utility==future risk

Off-tumour association: non-“pertinent” (‘secondary’ finding) -utility==future risk

Clinical ‘actionability’ of genes (==utility of a true germline finding)

Penetrance

Gene-level: High eg BRCA1/BRCA2, Intermediate eg CHEK2 1100delC

Variant –level (ATM V2424G)

Variant Pathogenicity: confidence

?Pathogenic (Class 5), ?Likely pathogenic (Class 4) ?VUS ?hot3-VUS

CONSIDERATIONS: SCIENTIFIC/CLINICAL

Workflow, Skillset of molecular pathology lab, turnaround time

Manpower, cost,

Germline interpretation-?individualised ‘ACMG’ scoring approach

?Delay tumour reporting ?Dual reporting

Timing:

Patient information-giving

Consent

Obtaining blood sample

?Upfront in all

?Two stage approach

?Tumour-type specific approach

CONSIDERATIONS: LOGISTIC







Meetings: 6

Data analyses: Mandelker+team, Turnbull

WORKING GROUP

1. 17,622 cases, paired tumour and blood sequencing using MSK-IMPACT panel

358 cases excluded: MSI-high, hyper-mutated, TMZ treated

METHODS I

TUMOUR TYPES ANALYSED Non-Small Cell Lung Cancer 2810 Germ Cell Tumor 390 Non-Hodgkin Lymphoma 72 Penile Cancer 9

Breast Cancer 2644 Thyroid Cancer 314 Small Bowel Cancer 63 Vaginal Cancer 8

Colorectal Cancer 1564 Head and Neck Cancer 295 Anal Cancer 49 Gestational Trophoblastic

Disease 6

Prostate Cancer 1207 Gastrointestinal Stromal

Tumor 233 Ampullary Carcinoma 48 Pheochromocytoma 5

Pancreatic Cancer 994 Bone Cancer 216 Thymic Tumor 45 Pineal Tumor 4

Glioma 941 Skin Cancer, Non-

Melanoma 203 Adrenocortical Carcinoma 44 Histiocytosis 3

Soft Tissue Sarcoma 680 Mesothelioma 180 Nerve Sheath Tumor 34 Hodgkin Lymphoma 3

Melanoma 665 Salivary Gland Cancer 168 Sex Cord Stromal Tumor 29 Leukemia 2

Bladder Cancer 587 Small Cell Lung Cancer 136 Sellar Tumor 25 Adenocarcinoma In Situ 1

Endometrial Cancer 569 Embryonal Tumor 130 Breast Sarcoma 24 Choroid Plexus Tumor 1

Hepatobiliary Cancer 551 Appendiceal Cancer 129 Miscellaneous Brain Tumor 15

Renal Cell Carcinoma 525 Uterine Sarcoma 129 Miscellaneous

Neuroepithelial Tumor 13

Esophagogastric Cancer 461 Gastrointestinal

Neuroendocrine Tumor 90 Retinoblastoma 12

Cancer of Unknown Primary 428 Cervical Cancer 87 Wilms Tumor 11

Ovarian Cancer 398 CNS Cancer 75 Mastocytosis 11

1. 17,622 cases, paired tumour and blood sequencing using MSK-IMPACT panel

358 cases excluded: MSI-high, hyper-mutated, TMZ treated

2. Cancer Susceptibility genes extracted from dual dataset

64 AD genes implicated in cancer susceptibility present on MSK IMPACT germline panel +1 AR

gene (MuTYH, as ACMG)

1,959,587 vars (somatic+germline)

3. Automated ‘pathogenicity pipeline’

Rare variants (MAF <0.01) 79,342 vars (somatic+germline)

Truncating if LOF gene and/or Pathogenic/LP on ClinVar 17,075 vars (somatic+germline)

Removal of low penetrance mutations in high penetrance genes (previously ‘pathogenic’

in ClinVar):

– APC (NM_000038.5) c.3920T>A (p.Ile1307Lys)

– VHL (NM_000551.3) c.598C>T (p.Arg200Trp)

– FH (NM_000143.3) c.1431_1433dupAAA (p.Lys477dup)

METHODS I

All tumours

Associated

tumours

Non-

associated

tumours

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(A) Application of serial filters to

MSK data on 65 genes for

16,322 tumours: number of

variants

1,997,499 1,959,587

Retained: MAF≤0.01 79,342 53,388 Retained: LP/P (ClinVar) or truncating 17,075 1,494 Retained: VAF ≥ 0.3

(SNV) or ≥ 0.2 (insdel)

All 9,222 1,442 2,904 454 6,305 983 HA-CSGs (AD) 6,141 677 2,259 326 3,882 351 SA-CSGs (AD)

2,372 213 539 37 1,820 176 Other (ie recessive or NA-

CSGs) 709 547 106 91 603 456 (B) Application of ESMO-PWG

recommendations for

gene/context/age criteria based

on 10% germline conversion:

number of variants

HA-CSGs (AD) all ages (18 genes) 851 615 410 300 441 315

age <30 (APC, RB1) 63 10 37 4 26 6 age <30, on-tumour only

(TP53) 59 7 59 7 n/a n/a

Total 973 632 506 311 467 321 SA-CSGs (AD) all ages, on tumour only

(BAP1, FH, FLN, POLE), 60 17 60 17 n/a n/a age <30, on tumour only

(NF1) 9 4 9 4 n/a n/a Total 69 21 69 21 n/a n/a

Grand Total 1,042 653 575 332 467 321

1. Retain only variants where VAF in tumour is

>20% (insertion/deletion)

>30% (SNV)

METHODS II

All tumours

Associated

tumours

Non-

associated

tumours

Tu

mour

dete

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Tu

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variants

1,997,499 1,959,587






recommendations for



number of variants



(TP53) 59 7 59 7 n/a n/a




Grand Total 1,042 653 575 332 467 321

PREMLINARY ANALYSIS

Variants in 65 CSGs (64 AD, 1 AR)

MAF <0.01

Path/LP (ClinVar)/truncating

VAF>0.3 (SNPs) VAF>0.2 insdels

9,222 variants (1,442 genuine germline)

METHODS III Assignation of tumour-gene combination as ‘associated’, ‘non-associated’ (germline)

Independently reviewed by 4 cancer clinical geneticists (≥2 support ‘associated’)

Assignation of genes by actionability

High Actionability: ACMG 25 CGSs (24 AD, 1 AR)+ (PALB2, RAD51C, RAD51D, BRIP1,

SDHA):

Standard Actionability : other dominant high penetrance CSGs in clinical usage

Questionable Actionability :

– intermediate penetrance,

– uncertainty of association of monoallelic variants with disease

(BARD1)

Threshold for triggering germline test

UK: 10% rate for detection of pathogenic mutation (NICE 2013;Familial Breast Cancer).

Only single fragment test…but also ‘additional aspects on process’

– ?just molecular test (ie ‘consent’+blood draw upfront)

– ?full genetics consultation+blood draw+molecular test

ALK

APC

ATM

BAP1

BARD1

BMPR1A

BRCA1

BRCA2

BRIP1

CDH1

CDK4

CDKN2A

CHEK2

DICER1

EPCAM

ERCC3

FH FLCN

HOXB13

HRAS

KIT

KRAS

MEN

1

MET

MITF

MLH1

MSH

2

MSH

6

MUTYH

NBN

NF1

NF2

NRAS

PALB2

PDGFRA

PMS2

POLE

PTCH1

PTEN

RAD50

RAD51B

RAD51C

RAD51D

RB1

RET

RUNX1

SDHA

SDHAF2

SDHB

SDHC

SDHD

SMAD3

SMAD4

SMARCA4

SMARCB1

STK11

SUFU

TERT

TGFBR1

TGFBR2

TMEM

127

TP53

TSC1

TSC2

VHL

HA-CSG N Y N N N Y Y Y Y N N N N N N N N N N N N N Y N N Y Y Y Y N N Y N Y N Y N N Y N N Y Y Y Y N Y Y Y Y Y N Y N N Y N N N N N Y Y Y Y

Penetrance H H I H H H H H H H H H I H H H H H I H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H

Robust for clinical implementation Y Y Y Y N Y Y Y Y Y Y Y Y Y N Y Y Y Y Y Y Y Y Y N Y Y Y Y N Y Y Y Y Y Y Y Y Y N Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

Adenocarcinoma In Situ

Adrenocortical Carcinoma

Ampullary Carcinoma

Anal Cancer

Appendiceal Cancer

Bladder Cancer

Bone Cancer

Breast Cancer

Breast Sarcoma

CNS Cancer

Cancer of Unknown Primary

Cervical Cancer

Colorectal Cancer

Embryonal Tumour

Endometrial Cancer

Oesophagogastric Cancer

GIST

Gastrointestinal NET

Germ Cell Tumour

Glioma

Head and Neck Cancer

Hepatobiliary Cancer

Histiocytosis

Melanoma

Mesothelioma

Miscellaneous Brain Tumour

NHL

NSCLC

Nerve Sheath Tumour

Ovarian Cancer

Pancreatic Cancer

Penile Cancer

Phaeochromocytoma

Pineal Tumour

Prostate Cancer

Renal Cell Carcinoma

Retinoblastoma

Salivary Gland Cancer

Sellar Tumour

Sex Cord Stromal Tumour

Skin Cancer

Small Bowel Cancer

Small Cell Lung Cancer

Soft Tissue Sarcoma

Thymic Tumour

Thyroid Cancer

Uterine Sarcoma

Vaginal Cancer

Wilms Tumour

neuro misc

Trophoblast





SDHA):





(BARD1)






HA-GENES (“ACMG+” LIST OF 30 GENES)

OFF TUMOUR:

→ 20 GENES

(19 AD+MUTYH)


ON TUMOUR:


OFF TUMOUR:


ON TUMOUR:


OFF TUMOUR:


ON TUMOUR:

VHL: ALL TUMOURS


OFF TUMOUR:

→ 20 GENES

(19 AD+MUTYH)


ON TUMOUR:

APC: ALL TUMOURS





SDHA):





(BARD1)






OTHER CANCER SUSCPTIBILITY GENES: ON TUMOUR

METHODS V: ARE WE MISSING ANY GROUPS

Any idiosyncratic combinations of tumour and gene for which germline attribution is

high?

Examined all combinations

Are there subgroups of patients defined by age of cancer onset for which germline

attribution is high?

Are there genes for which there are a set of particular mutations for which germline


TUMOUR-GENE

COMBINATIONS

METHODS V: ARE WE MISSING ANY GROUPS


high?






PREMLINARY ANALYSIS:

TUMOURS ARISING AGE <30

Variants in 65 CSGs (64 AD, 1 AR)

MAF <0.01

Path/LP (ClinVar)/truncating

VAF>0.3 (SNPs) VAF>0.2 insdels

All (on tumour AND off tumour)

Examining HA genes


ON TUMOUR:

AGE<30: ALL GENES; ON TUMOUR

Examining SA

genes

And ?HA too

METHODS IV: ARE WE MISSING ANY GROUPS


high?






Variant Total tumour-observed variants Germline origin Somatic origin

c.527G>T p.C176F 17 1 16

c.665delC p.P222Rfs*25 2 1 1

c.713G>C p.C238S 2 1 1

c.422G>A p.C141Y 6 1 5

c.818G>C p.R273P 4 1 3

c.916C>T p.R306* 32 2 30

c.273G>A p.W91* 10 2 8

c.743G>A p.R248Q 103 2 101

c.742C>T p.R248W 89 2 87

c.586C>T p.R196* 51 1 50

c.1009C>T p.R337C 17 1 16

c.638G>A p.R213Q 8 1 7

c.818G>A p.R273H 104 1 103

c.536A>G p.H179R 17 1 16

c.919+1G>C

p.X307_splice 2 1 1

c.524G>A p.R175H 157 1 156

c.733G>A p.G245S 52 2 50

c.542G>A p.R181H 3 2 1

c.403T>C p.C135R 5 1 4

c.844C>G p.R282G 7 1 6

TP53: ANALYSIS

BY MUTATION

1) Germline-focused analysis should be performed routinely on tumour-only sequencing (where

there is full sequencing of genes which are involved in cancer susceptibility)

2) Germline follow-up should be undertaken when ≥10% likelihood of variant in HA/SA gene

being germline in origin

3) Analyse only for Class 4/5 germline variants is reccommended

4) Analyse only genes with strong evidence for AD cancer susceptibility (not BARD1, NBN)

5) Intermediate penetrance genes (CHEK2, ATM) do not need to be included.

6) An automated LOF+ClinVar pipeline can be applied

SUMMARY: RECOMMENDATIONS (GENERAL)

1) HA-CSGs (on and off tumour): 19 +1 genesBMPR1A, BRCA1, BRCA2, BRIP1, MLH1,

MSH2, MSH6, MUTYH*, PALB2, PMS2, RAD51C, RAD51D, RET, SDHA, SDHAF2, SDHB,

SDHC, SDHD, TSC2, VHL**,

2) SA-CSGs (on tumour only): FH, FLCN, BAP1, POLE

3) Analysis if tumour arose <30

1) On and off tumour: RB1, APC

2) On tumour: NF1, TP53***

1. Ahead of confirmatory analysis in germline sample, patients should be provided with

information and offered option to opt-in or opt-out of analysis of germline sample

SUMMARY: RECOMMENDATIONS (SPECIFIC)

MuTYH only taken forwards if biallelic mutations present *VHL analysis not recommended in renal cancers, *** TP53 analysis not recommended in brain cancers

SUMMARY: RECOMMENDATIONS (CONSENT)

All tumours

Associated

tumours

Non-

associated

tumours

Tu

mour

dete

cte

d

Tru

e

germ

line

Tu

mour

dete

cte

d

Tru

e

germ

line

Tu

mour

dete

cte

d

Tru

e

germ

line




variants

1,997,499 1,959,587






recommendations for



number of variants



(TP53) 59 7 59 7 n/a n/a




Grand Total 1,042 653 575 332 467 321

In 17,622 large tumour panels:

ON-TUMOUR: 575 germline tests 332 true germline (57%)

OFF TUMOUR: 487 germline tests 321 true germline (66%)

COMBINED: 1042 germline tests (6.7%) 653 true germline (63%)

IMPACT

Working Group:







ESMO: Svetlana Jezdic, Fabrice Andre, Jean-Yves Douillard

Expert Genetics Panel: Drs Helen Hanson, Katie Snape, Anjana Kulkani

Painless access to MSK data: Prof Marc Ladyani

ACKNOWLEDGEMENTS

Documents

INDICATIONS FOR GERMLINE TESTING FROM TUMOUR-ONLY … · Neuroendocrine Tumor 90 Retinoblastoma 12 Cancer of Unknown Primary 428 Cervical Cancer 87 Wilms Tumor 11 Ovarian Cancer 398