Tumor Mutational Burden (TMB)...Tumor Mutational Burden (TMB) or Tumor Mutation Load (TML) Somatic...

Carico mutazionale tumorale

Caterina Fumagalli

Istituto Europeo di Oncologia - Milano

Metastatic MelanomaHodi NEJM 2010

Advanced NSCLCBorghaei NEJM 2015

Advanced Urothelial CarcinomaBellmunt NEJM 2017

15%-20% responders

Advanced Renal Cell CarcinomaMotzer NEJM 2018

Immunotherapy improves Overall Survival in several diseases

Immune checkpoint inhibitors have changed the treatment paradigm for a

variety of cancers, but not all patients will respond to immunotherapies.

Biomarker Need for patients selection

Therapy

Immunotherapy Responders

From Chen DS, Mellman I «Oncology Meets Immunology:The Cancer-Immunity Cycle» Immunity 2013, 1-10

How to predict immunotherapy response? Looking for biomarker

TMB or TML: total number of somatic/acquired mutations per

coding area of a tumor genome (Mut/Mb)

The number of mutations can vary across different tumor types.

Tumor Mutational Burden (TMB) or Tumor Mutation Load (TML)

Somatic mutation frequencies observed in exomes from 3,083 tumor-normal pairs

From Lawrence MS et al Nature 2013, 499:214–218

Tumor mutation frequencies & different tumor types

Distinct mechanisms of DNA mutation:

* MMR deficiency

* Exposure to environmental mutagens

(tobacco smoke and UV light)

* Virus – associated tumors

Tumor Mutational Burden (TMB) ….

What does it means?

Nonsynonimous mutations have the potential to generate neoantigens recognized by

the host immune system, leading to an antitumor immune response.

From Braun DA et al. Clin Cancer Res 2016 22(23): 5642-5650

TMB & neoantigen formation

From Braun DA et al. Clin Cancer Res 2016 22(23): 5642-5650

TMB & neoantigen formation

From Sharabi et al The Oncologist 2017, 22:631–637

Tumors with high mutation burden have the potential to generate a larger number of neoantigens,

making them more immunogenic.

High vs Low Tumor Mutational Burden (TMB)

Tumor Mutational Burden (TMB)

&

Immunotherapy response

Rizvi et al «Mutational Landscape determines sensitivity to PD-1 blockade in non-smallcell lung cancer» Science 2015

Snyder et al “Genetic Basis for Clinical Response to CTLA-4 Blockade in Melanoma” NEJM2014

TMB predictive biomarker of immunotherapy

From Yarchoan M et al. N ENGL J MED 377:25, 2017

TMB predictive biomarker of immunotherapy

TMB could be an optimal biomarker but

how to implement TMB testing in

routine clinical practice?

TMB evaluation: Clinical practise

Progenitor studies: WES (Whole Exome sequencing)

BUT

If TMB becomes a marker for use in routine clinical practice, it is more likely to be determined by NGS rather than by WES

Targeted NGS panels

76 cancer-genes 160 cancer-genes 195 cancer-genes

315 cancer-genes 641 cancer-genes

Campesato FL et al «Comprehensive cancer-gene panels can be used to estimate mutational load

and predict clinical benefit to PD-1 blockade in clinical practice” Oncotarget 2015, Vol. 6, No. 33

How much «Targeted»?

TMB testing Panels

Panel

Diagnostic-

Use/FDA

approved

Chemistry # Genes Megabases Threshold Result Study

FoundationOne

CDx - RocheYes

Hybrid-

capture-

based NGS

324 1,2 Mb YES

HIGH= ≥ 20 MUT/Mb

INTERMEDIATE = 6-19 MUT/Mb

LOW = 1-5 MUT/Mb

Goodman AM Mol Canc Ther 2017

Hel lmann MD NEJM 2018

CGP+ - Agilent

SureSelect XT -

Caris Molecular

Intelligence

No

Hybrid-

capture-

based NGS

592 1,4 Mb YESHIGH= ≥ 17 MUT/Mb

LOW = < 17 MUT/Mb

Gatal ica Z Eur Journal Canc 2018

Vandervalde A Cancer Med 2018

MSK-IMPACT Yes

Hybrid-

capture-

based NGS

468 1,22 Mb NO MUT/Mb Rizvi H JCO 2018

TruSight Tumor

170 - ILLUMINANo

Hybrid-

capture-

based NGS

170 0,524 Mb NO MUT/Mb In silico analyses

Oncomine™

Tumor Mutation

Load Assay -

ThermoFisher

Scientific

NoAmplicon-

based NGS409 1,7 Mb NO MUT/Mb In silico analyses

Déjà-vu Harmonization Study

Panel

Diagnostic-

Use/FDA

approved


FoundationOne

CDx - RocheYes

Hybrid-

capture-

based NGS

324 1,2 Mb YES

HIGH= ≥ 20 MUT/Mb


LOW = 1-5 MUT/Mb



CGP+ - Agilent

SureSelect XT -

Caris Molecular

Intelligence

No

Hybrid-

capture-

based NGS


LOW = < 17 MUT/Mb



MSK-IMPACT Yes

Hybrid-

capture-

based NGS


TruSight Tumor

170 - ILLUMINANo

Hybrid-

capture-

based NGS


Oncomine™

Tumor Mutation

Load Assay -

ThermoFisher

Scientific

NoAmplicon-


TMB tests differences - # of genes

Panel

Diagnostic-

Use/FDA

approved


FoundationOne

CDx - RocheYes

Hybrid-

capture-

based NGS

324 1,2 Mb YES

HIGH= ≥ 20 MUT/Mb


LOW = 1-5 MUT/Mb



CGP+ - Agilent

SureSelect XT -

Caris Molecular

Intelligence

No

Hybrid-

capture-

based NGS


LOW = < 17 MUT/Mb



MSK-IMPACT Yes

Hybrid-

capture-

based NGS


TruSight Tumor

170 - ILLUMINANo

Hybrid-

capture-

based NGS


Oncomine™

Tumor Mutation

Load Assay -

ThermoFisher

Scientific

NoAmplicon-


TMB test differences - # of Mut/Mb (cut off?)

“ As the cutoff used to dichotomize TMB between low and high

increases, the outcome improves in a linear fashion, favoring

the TMB high group”.

Mol Cancer Ther; 16(11) November 2017

“Odds ratio (OR) of DCB with increasing cut points of TMB. 25th (OR,

1.75), 50th (OR, 2.02), 75th (OR, 2.06), and 90th (OR, 3.24)

percentiles. The 0 percentile (white bar) is shown for reference of all

patients (default OR, 1). The odds of DCB increase significantly above

the 50th percentile of TMB”.

J Clin Oncol. 2018 Mar 1;36(7):633-641

TMB test differences - # of Mut/Mb (cut off?)

Panel: FoundationOne assayHigh TMB ≥ 10 mut/MbLow TMB < 10 mut/Mb

Hellmann MD et al «Nivolumab plus ipilimumab in

lung cancer with a high tumor mutational burden»

NEJM, April 2018

Panel: FoundationOne assayHigh TMB ≥ 20 mut/MbIntermediate-Low TMB

Goodman AM et al «Tumor Mutational Burden as an

independent predictor of response to immunotherapy in diverse

cancers»

Mol Cancer Ther, November 2017

▪ Type of mutation

• SNV/indel

• synonymous/nonsynonymous

• coding/non coding regions

TMB tests differences - algorithm

▪ Normal tissue for comparison / database annotation filtering

Panel

Diagnostic-

Use/FDA

approved


FoundationOne

CDx - RocheYes

Hybrid-

capture-

based NGS

324 1,2 Mb YES

HIGH= ≥ 20 MUT/Mb


LOW = 1-5 MUT/Mb



CGP+ - Agilent

SureSelect XT -

Caris Molecular

Intelligence

No

Hybrid-

capture-

based NGS


LOW = < 17 MUT/Mb



MSK-IMPACT Yes

Hybrid-

capture-

based NGS


TruSight Tumor

170 - ILLUMINANo

Hybrid-

capture-

based NGS


Oncomine™

Tumor Mutation

Load Assay -

ThermoFisher

Scientific

NoAmplicon-


Comprehensive cancer-related gene profiling

TMB tests differences – NGS panels

TMB dedicated panel

TMB tests differences: Outsourced / In-House

"OUTSOURCED"

- Laboratory instrument implementation

- Dedicated space

- Trained Staff (Expertise/Resources)

- Cost

“IN-HOUSE"

Panel

Diagnostic-

Use/FDA

approved


FoundationOne

CDx - RocheYes

Hybrid-

capture-

based NGS

324 1,2 Mb YES

HIGH= ≥ 20 MUT/Mb


LOW = 1-5 MUT/Mb



CGP+ - Agilent

SureSelect XT -

Caris Molecular

Intelligence

No

Hybrid-

capture-

based NGS


LOW = < 17 MUT/Mb



MSK-IMPACT Yes

Hybrid-

capture-

based NGS


TruSight Tumor

170 - ILLUMINANo

Hybrid-

capture-

based NGS


Oncomine™

Tumor Mutation

Load Assay -

ThermoFisher

Scientific

NoAmplicon-


TMB testing Panels

Panel

Diagnostic-

Use/FDA

approved


FoundationOne

CDx - RocheYes

Hybrid-

capture-

based NGS

324 1,2 Mb YES

HIGH= ≥ 20 MUT/Mb


LOW = 1-5 MUT/Mb



CGP+ - Agilent

SureSelect XT -

Caris Molecular

Intelligence

No

Hybrid-

capture-

based NGS


LOW = < 17 MUT/Mb



MSK-IMPACT Yes

Hybrid-

capture-

based NGS


TruSight Tumor

170 - ILLUMINANo

Hybrid-

capture-

based NGS


Oncomine™

Tumor Mutation

Load Assay -

ThermoFisher

Scientific

NoAmplicon-


Issue: Harmonization

TMB_IEO experience: preliminary data

Panel

Diagnostic-

Use/FDA

approved


FoundationOne

CDx - RocheYes

Hybrid-

capture-

based NGS

324 1,2 Mb YES

HIGH= ≥ 20 MUT/Mb


LOW = 1-5 MUT/Mb



CGP+ - Agilent

SureSelect XT -

Caris Molecular

Intelligence

No

Hybrid-

capture-

based NGS


LOW = < 17 MUT/Mb



MSK-IMPACT Yes

Hybrid-

capture-

based NGS


TruSight Tumor

170 - ILLUMINANo

Hybrid-

capture-

based NGS


Oncomine™

Tumor Mutation

Load Assay -

ThermoFisher

Scientific

NoAmplicon-


TMB Assay & Samples

Panel

Diagnostic-

Use/FDA

approved


FoundationOne

CDx - RocheYes

Hybrid-

capture-

based NGS

324 1,2 Mb YES

HIGH= ≥ 20 MUT/Mb


LOW = 1-5 MUT/Mb



CGP+ - Agilent

SureSelect XT -

Caris Molecular

Intelligence

No

Hybrid-

capture-

based NGS


LOW = < 17 MUT/Mb



MSK-IMPACT Yes

Hybrid-

capture-

based NGS


TruSight Tumor

170 - ILLUMINANo

Hybrid-

capture-

based NGS


Oncomine™

Tumor Mutation

Load Assay -

ThermoFisher

Scientific

NoAmplicon-

based NGS409 1,7 Mb NO MUT/Mb In silico analysesRUO

Library & Chip preparation: Fully Automated, starting from 20 ng FFPE DNA

Specimens (n=32): 16 Non-Small Cell Lung Cancers

11 Colorectal Cancers

5 Breast Cancers

Failure rate = 1/32 cases (3.1%)

TMB_IEO experience:Median Mutations per Mb

NSCLC = 10.75 mut/Mb CRC = 20.02 mut/Mb BC = 9.83 mut/Mb

TMB_IEO experience:Range Mutations per Mb

NSCLC = 10.75 mut/Mb CRC = 20.02 mut/Mb

4.33 – 28.65 mut/Mb 7.35 – 66.17 mut/Mb 5.51-17.32 mut/Mb

BC = 9.83 mut/Mb

16 NSCLC• Sample type

- TBNA smears: 4- Biopsies: 8- Surgical resection specimens: 4

• PD-L1 status- >50%: 15- NA: 1

• Molecular status- WT: 7- Molecular alteration: 9

TMB_IEO experience: NSCLC

NSCLC MUT= 9.84 mut/Mb NSCLC WT= 11.05 mut/Mb

▪ 11 CRC

Microsatellite analysis

- MSI-HIGH: 4

- MSI-LOW: 1 (failed analysis)

- MSS : 6

▪ 5 Breast cancers:

- TNBC: 4

- NEG ER/PGR HER2 3+>95%: 1

TMB_IEO experience:CRC & Breast Cancer

TMB_IEO experience:

NSCLC = 10.75 mut/Mb

CRC = 20.02 mut/Mb

BC = 9.83 mut/MbMSI-HIGH CRC 52.61 mut/Mb

MSS CRC 10.45 mut/Mb

TMB & other biomarkers of

immunotherapy response

TMB did not correlate with PD-L1 expression: “both

variables had similar predictive capacity. The

incorporation of both TMB and PD-L1 expression into

multivariable predictive models should result in greater

predictive power” Rizvi H et al JCO 2018 , 36(7): 633-

641

TMB & PD-L1 & MSI: “Among 11348 pts, only 0.6% of

the cases were positive for all three markers”

Vandervalde A et al Cancer Medicine January 2018

TMB & other biomarkers

Given the complexity of the immune response and tumor biology,

different biomarkers could better predict the response to

immunotherapies?

…. What about the clinical point of view?

Future: Combined biomarkers strategies?

…

Massimo Barberis

Gianluca De MarzoElena Guerini-Rocco

Paolo LopedoteAlberto RanghieroAlessandra Rappa

Mila SchiaviFrancesco Spinelli

Tania TamagniDavide Vacirca

Thank You

Molecular Diagnostics UnitDivision of Pathology

Hellmann MD et al «Nivolumab plus ipilimumab in lung

cancer with a high tumor mutational burden»

NEJM, April 2018

❖ Quale è il reale vantaggio (se pensi ci sia) del "marcatore” da tedescritto rispetto agli altri due

* Maggiore correlazione con la risposta allaterapia rispetto agli altri marcatori?

* In fase di studio anche su liquid biopsy

Gandara DR et al «Blood-based tumor mutational

burden as a predictor of clinical benefit in non-small-cell

lung cancer patients treated with atezolizumab»

Nat Med. 2018 Aug 6. doi: 10.1038/s41591-018-0134-3.

* Mancanza di validazione: In attesa degli studi di armonizzazione tra i diversi test TMB e

piattaforme NGS

* Processazione di più campioni contemporaneamente (test «in house»):

8 – 16 campioni contemporaneamente

❖ Quale è il principale difetto del “marcatore” da te descritto?

Panel

Diagnostic-

Use/FDA

approved


FoundationOne

CDx - RocheYes

Hybrid-

capture-

based NGS

324 1,2 Mb YES

HIGH= ≥ 20 MUT/Mb


LOW = 1-5 MUT/Mb



CGP+ - Agilent

SureSelect XT -

Caris Molecular

Intelligence

No

Hybrid-

capture-

based NGS


LOW = < 17 MUT/Mb



MSK-IMPACT Yes

Hybrid-

capture-

based NGS


TruSight Tumor

170 - ILLUMINANo

Hybrid-

capture-

based NGS


Oncomine™

Tumor Mutation

Load Assay -

ThermoFisher

Scientific

NoAmplicon-


❖ Quale è il costo indicativo OGGI per ottenere il risultato del test da te descritto?

700 - 1000 euro«In house»: solo test, senza personale,ottimizzando la seduta con ilmassimo di campioni processaticontemporaneamente

3000 – 4000 euro«Service»

* Campioni FFPE: 20 -40 ng DNA

* «Service»: Tessuto FFPE (blocchetto o sezioni con minimo di volume/cellularità

tumorale)

❖ Quanto materiale (e quale materiale) ti serve per eseguire il testda te descritto nella tua relazione?

* «Service»: TAT < 14 gg

* «In house»:

❖ Quale è il turnaround time per ottenere il risultato del test da tedescritto?

Accession

Material received

Pathologist review

DNA extraction

Analysis (run)

Analysis (data)

Report

- Considerare i tempi pre-test

- Processare più campioni contemporaneamente

❖ E’ indubbio che tutti noi in questa sessione veniamo da centri“selezionati”, ma ritieni che il test da te descritto sia OGGIfattibile in modo adeguato e con le caratteristiche esposte inquesta sessione in tutti i centri italiani?

Lack of harmonization of TMB tests across the platforms & definition of clinical utility

- Dedicated space/Laboratory instrument implementation

- Expertise/ resources

- Number of test requested Cost / Tornaround Time

"TMB is provocative and warrants further study, standardization, and prospective testing" "TMB is NOT ready for use in patient care“David L. Rimm from “TMB as New Biomarker in NSCLC -- Ready for Clinic?” - Medscape - Apr 17, 2018.

Tumor Mutational Burden (TMB)...Tumor Mutational Burden (TMB) or Tumor Mutation Load (TML) Somatic...

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