1. Validation & Tumour Board – GM GMC Experience
Andrew Wallace
04/06/2018
Genomic Diagnostics Laboratory
St. Mary’s Hospital, Manchester
First I’d like to tell you about the process being followed when results from cancer cases are released by GEL to the GMCs, then illustrate with a couple of examples how the process has worked and what we have found

2. Rate of Data Return from GEL is Challenging
Slow initially
Rapidly increasing
133 cases returned to date
Includes 5 urgent compassionate cases
Tumour type No
Lung 45
CRC 22
Endometrial 17
Renal 15
Ovarian 14
Breast 6
Sarcoma 5
Melanoma 3
Paediatric 2
Other 4
TOTAL
133

3. Managing GEL WGS Data is Challenging
In preparation for the 100kGP Tumour Board need
WGS data
Variant interpretations
Demographic details
Pathology reports
SOC test results
Current clinical state
Initially used MS-Word to present results
Mail merge from export of demographic data from Lab Key and Themis
Process SNV data downloaded as csv from IP in MS Excel then copy & paste

4. Moved to MS Excel for Tumour Boards
Single sheet for each patient
Workbook(s) for each Tumour Board
Pasting locations
exported clinical and demographic data from LabKey and Themis
csv files with variants from interpretation portal
Embedded files for Path reports etc

5. CNVs, SVs, Tumour Burden & Signatures
Routinely mine the WGS data for non-’domained’ variants
Has been useful e.g.
SVs
Indication of possible fusion genes e.g. RET, ALK, ROS1
CNVs
Allele loss for mutations in tumour suppressor genes
Gene amplification at relevant loci e.g. ERBB2, MET
Mutation signatures
HRD associated pattern accompanying HRR pathway gene mutations
Tumour mutation burden
Potential for response to immunotherapies
Record on spreadsheets
Specific SVs and CNVs depending on organ of origin

6. GM GMC Tumour Board Fixed timetable – every 2 weeks
Postpone if no cases
60-90mins
Group cases for discussion by tumour type
10-14 cases discussed each session
Specific lead oncologists & pathologists invited according to tumour type
Urgent & compassionate cases listed at next available board
Cases can be and are re-listed
Dial in & webex available

7. Recording Outcomes Interim MS-Access database records
Data return
WGS results
MTB discussions and outcomes
Variant confirmations
Manual entry
Web based 100k dashboard application in development
Data download from CIP-API
Data upload to GEL (subject to permission)

8. Variant Confirmations
More frequent than expected!
Mixture of
clinically actionable
possibly informative (clinical trial)
precautionary (exclude germline mosaicism)
27 samples with 32 variant confirmations scheduled in total
29/32 variant confirmations using NGS panels 13 2
All variants confirmed
One case additional finding (polyclonal) KRAS variant 3 4

9. Reporting
Issuing reports (positive and negative) on all Tumour Board reviewed cases
Report to:
Pathologist for integration into pathology report
Oncologist where appropriate (e.g. patient under treatment)
Local requirement to include in Notes all Domain 1 variants with ACMG class 3-5 with an option for confirmation
Report includes ‘pertinent‘ germline status

10. GM GMC Example Case 1 Compassionate urgent WGS case 15yo Female
Metastatic disease (lung, bone, liver & pancreas)
Likely pancreatic primary
Pathology of liver biopsies showed a poorly differentiated neuroendocrine carcinoma
Excluded Ewing’s sarcoma, desmoplastic small round cell tumour, neuroblastoma, rhabdomysosarcoma, Wilms tumour, melanoma & lymphoma
Patient consented 25/10/18
DNA submitted to GEL 13/12/2017
Results released to GM GMC 30/04/2018

11. GM GMC Example Case 1 No domain 1 variants 7 domain 2 variants
A high proportion of variants are del/dupT
196/236 in homopolymers
77/196 delT – 93/196 dupT
10/96 delA – 13/196 dupA
7.5x more somatic indels than SVs
229 domain 3 variants

12. GM GMC Example Case 1 Discussed at Tumour Board on 03/05/18
30% of paediatric neuroendocrine tumours associated with hereditary cancer syndromes1
Neuroendocrine tumours occasionally arise in intestinal adenomas in FAP2
Discussed at Tumour Board on 03/05/18
Confirmed by Sanger 10/05/18
Reported 17/05/18
No FH of bowel cancer
O/E patient has bilateral CHRPES
Referred for colonoscopy
Parents and sister scheduled for Genetic Counselling
1J. Gaal and R. R. de Krijger, “Neuroendocrine tumors and tumor syndromes in childhood,” Pediatric and Developmental Pathology, vol. 13, no. 6, pp. 427–441, 2010. 
2 Estrella et al (2014) Low-grade neuroendocrine tumors arising in intestinal adenomas: evidence for alterations in the adenomatous polyposis coli/β-catenin pathway45, 10, 2051–2058

13. Validation & Tumour Board – GM GMC Experience
Andrew Wallace
04/06/2018
Genomic Diagnostics Laboratory
St. Mary’s Hospital, Manchester
First I’d like to tell you about the process being followed when results from cancer cases are released by GEL to the GMCs, then illustrate with a couple of examples how the process has worked and what we have found

14. GM GMC Example Case 2
55y patient with high grade serous ovarian adenocarcinoma
Patient relapsed May 2017 currently on chemotherapy
BRCA1/2 germline normal
WGS identified 7 domain 1 variants
Following GMC review 1 classified as Tier 1A & 4 as Tier 2C/D
Tier 1A BRCA1 c.1059G>A p.(Trp353Ter)
Pathogenic nonsense mutation
Tier 2C/D NF1, TP53, PIK3CA & CTNNA3
NF1 & TP53 commonly mutated together in OvCa
Released the results in advance of the Tumour board to the Oncologist who asked for immediate validation of the BRCA1 finding. We showed that this was somatic (only in tumour)
Interestingly NF1 and TP53 mutations usually occur together in ovarian cancer
BRCA1 c.1059G>A p.(Trp353Ter) confirmed by Sanger analysis
Bi-allelic BRCA inactivation queried at Tumour Board

15. GM GMC Example Case 2 Signature 3 present in ~30% of ovarian cancers
We are able to search the WGS report file for specific SVs of interest
Structural variants (copy number variants and genomic re-arrangements) are reported but the analytic pipeline is not validated
WGS mutation profile matched to one of 30 mutation signatures
Mutation signature 3 present & consistent with Homologous Recombination Deficiency
Signature 3 present in ~30% of ovarian cancers

16. GM GMC Example Case 2 Circos plot - genome level overview
BRCA1
Currently undertaking BRCA1 MLPA analysis to confirm the likely deletion
Method of displaying genome level information including structural variants (SVs) and copy number variants (CNVs)
Shows a large number of translocations, consistent with HRD
Patient now enrolled in MOLTO clinical trial for PARPi therapy

17. GM GMC Example Case 3
65y patient with low grade ovarian adenocarcinoma
WGS identified 4 domain 1 variants
Following GMC review 3 classified as Tier 3/Tier 4
One KRAS c.34G>C p.Gly12Arg known activating mutation classified as Tier 2C
Outcome of discussion at Tumour Board - patient may be eligible for LOGS study hence request to confirm the KRAS variant

18. Confirmation of KRAS variant
Sample run with 24 gene NGS panel used for SOC testing
Second pathogenic KRAS mutation present!
c.35G>T
p.Gly12Val
c.34G>C
p.Gly12Arg
Variants do not occur together – in different tumour clones (or on different alleles)

19. Review of WGS data by GEL
KRAS c.35G>T p.(Gly12Val) present
4/148 reads (3%)
Below LOD for calling for WGS pipeline
Variants on different reads
WGS less sensitive than GMC NGS assay – lower coverage depth

20. Conclusions 100kGP WGS data being generated on tumour DNAs
Clinically relevant findings being identified outside of standard of care
GM-GMC is developing processes to manage the data flow and reporting
WGS evolving not (yet) ready to replace standard of care testing

21. Validation & Tumour Board – GM GMC Experience
Andrew Wallace
22/09/2017
Genomic Diagnostics Laboratory
St. Mary’s Hospital, Manchester