Carico mutazionale tumorale
Caterina Fumagalli
Istituto Europeo di Oncologia - Milano
Metastatic Melanoma Hodi NEJM 2010
Advanced NSCLC Borghaei NEJM 2015
Advanced Urothelial Carcinoma Bellmunt NEJM 2017
15%-20% responders
Advanced Renal Cell Carcinoma Motzer 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-small cell lung cancer» Science 2015
Snyder et al “Genetic Basis for Clinical Response to CTLA-4 Blockade in Melanoma” NEJM 2014
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 - Roche Yes
Hybrid- capture- based NGS
324 1,2 Mb YES
HIGH= ≥ 20 MUT/Mb INTERMEDIATE = 6-19 MUT/Mb
LOW = 1-5 MUT/Mb
Goodma n AM Mol Ca nc Ther 2017 Hel l ma nn MD NEJM 2018
CGP+ - Agilent SureSelect XT - Caris Molecular Intelligence
No
Hybrid- capture- based NGS
592 1,4 Mb YES HIGH= ≥ 17 MUT/Mb LOW = < 17 MUT/Mb
Ga tal i ca Z Eur Journa l Ca nc 2018 Va nderva l de A Ca ncer Med 2018
MSK-IMPACT Yes
Hybrid- capture- based NGS
468 1,22 Mb NO MUT/Mb Ri zvi H JCO 2018
TruSight Tumor
170 - ILLUMINA No
Hybrid- capture- based NGS
170 0,524 Mb NO MUT/Mb In silico a na l ys es
Oncomine™
Tumor Mutation Load Assay - ThermoFisher
Scientific
No Amplicon-
based NGS 409 1,7 Mb NO MUT/Mb In silico a na l ys es
Déjà-vu Harmonization Study
Panel
Diagnostic- Use/FDA approved
Chemistry # Genes Megabases Threshold Result Study
FoundationOne
CDx - Roche Yes
Hybrid- capture- based NGS
324 1,2 Mb YES
HIGH= ≥ 20 MUT/Mb INTERMEDIATE = 6-19 MUT/Mb
LOW = 1-5 MUT/Mb
Goodma n AM Mol Ca nc Ther 2017 Hel l ma nn MD NEJM 2018
CGP+ - Agilent SureSelect XT - Caris Molecular Intelligence
No
Hybrid- capture- based NGS
592 1,4 Mb YES HIGH= ≥ 17 MUT/Mb LOW = < 17 MUT/Mb
Ga tal i ca Z Eur Journa l Ca nc 2018 Va nderva l de A Ca ncer Med 2018
MSK-IMPACT Yes
Hybrid- capture- based NGS
468 1,22 Mb NO MUT/Mb Ri zvi H JCO 2018
TruSight Tumor
170 - ILLUMINA No
Hybrid- capture- based NGS
170 0,524 Mb NO MUT/Mb In silico a na l ys es
Oncomine™
Tumor Mutation Load Assay - ThermoFisher
Scientific
No Amplicon-
based NGS 409 1,7 Mb NO MUT/Mb In silico a na l ys es
TMB tests differences - # of genes
Panel
Diagnostic- Use/FDA approved
Chemistry # Genes Megabases Threshold Result Study
FoundationOne
CDx - Roche Yes
Hybrid- capture- based NGS
324 1,2 Mb YES
HIGH= ≥ 20 MUT/Mb INTERMEDIATE = 6-19 MUT/Mb
LOW = 1-5 MUT/Mb
Goodma n AM Mol Ca nc Ther 2017 Hel l ma nn MD NEJM 2018
CGP+ - Agilent SureSelect XT - Caris Molecular Intelligence
No
Hybrid- capture- based NGS
592 1,4 Mb YES HIGH= ≥ 17 MUT/Mb LOW = < 17 MUT/Mb
Ga tal i ca Z Eur Journa l Ca nc 2018 Va nderva l de A Ca ncer Med 2018
MSK-IMPACT Yes
Hybrid- capture- based NGS
468 1,22 Mb NO MUT/Mb Ri zvi H JCO 2018
TruSight Tumor
170 - ILLUMINA No
Hybrid- capture- based NGS
170 0,524 Mb NO MUT/Mb In silico a na l ys es
Oncomine™
Tumor Mutation Load Assay - ThermoFisher
Scientific
No Amplicon-
based NGS 409 1,7 Mb NO MUT/Mb In silico a na l ys es
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 assay High TMB ≥ 10 mut/Mb
Low 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 assay High TMB ≥ 20 mut/Mb
Intermediate-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
Chemistry # Genes Megabases Threshold Result Study
FoundationOne
CDx - Roche Yes
Hybrid- capture- based NGS
324 1,2 Mb YES
HIGH= ≥ 20 MUT/Mb INTERMEDIATE = 6-19 MUT/Mb
LOW = 1-5 MUT/Mb
Goodma n AM Mol Ca nc Ther 2017 Hel l ma nn MD NEJM 2018
CGP+ - Agilent SureSelect XT - Caris Molecular Intelligence
No
Hybrid- capture- based NGS
592 1,4 Mb YES HIGH= ≥ 17 MUT/Mb LOW = < 17 MUT/Mb
Ga tal i ca Z Eur Journa l Ca nc 2018 Va nderva l de A Ca ncer Med 2018
MSK-IMPACT Yes
Hybrid- capture- based NGS
468 1,22 Mb NO MUT/Mb Ri zvi H JCO 2018
TruSight Tumor
170 - ILLUMINA No
Hybrid- capture- based NGS
170 0,524 Mb NO MUT/Mb In silico a na l ys es
Oncomine™
Tumor Mutation Load Assay - ThermoFisher
Scientific
No Amplicon-
based NGS 409 1,7 Mb NO MUT/Mb In silico a na l ys es
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
Chemistry # Genes Megabases Threshold Result Study
FoundationOne
CDx - Roche Yes
Hybrid- capture- based NGS
324 1,2 Mb YES
HIGH= ≥ 20 MUT/Mb INTERMEDIATE = 6-19 MUT/Mb
LOW = 1-5 MUT/Mb
Goodma n AM Mol Ca nc Ther 2017 Hel l ma nn MD NEJM 2018
CGP+ - Agilent SureSelect XT - Caris Molecular Intelligence
No
Hybrid- capture- based NGS
592 1,4 Mb YES HIGH= ≥ 17 MUT/Mb LOW = < 17 MUT/Mb
Ga tal i ca Z Eur Journa l Ca nc 2018 Va nderva l de A Ca ncer Med 2018
MSK-IMPACT Yes
Hybrid- capture- based NGS
468 1,22 Mb NO MUT/Mb Ri zvi H JCO 2018
TruSight Tumor
170 - ILLUMINA No
Hybrid- capture- based NGS
170 0,524 Mb NO MUT/Mb In silico a na l ys es
Oncomine™
Tumor Mutation Load Assay - ThermoFisher
Scientific
No Amplicon-
based NGS 409 1,7 Mb NO MUT/Mb In silico a na l ys es
TMB testing Panels
Panel
Diagnostic- Use/FDA approved
Chemistry # Genes Megabases Threshold Result Study
FoundationOne
CDx - Roche Yes
Hybrid- capture- based NGS
324 1,2 Mb YES
HIGH= ≥ 20 MUT/Mb INTERMEDIATE = 6-19 MUT/Mb
LOW = 1-5 MUT/Mb
Goodma n AM Mol Ca nc Ther 2017 Hel l ma nn MD NEJM 2018
CGP+ - Agilent SureSelect XT - Caris Molecular Intelligence
No
Hybrid- capture- based NGS
592 1,4 Mb YES HIGH= ≥ 17 MUT/Mb LOW = < 17 MUT/Mb
Ga tal i ca Z Eur Journa l Ca nc 2018 Va nderva l de A Ca ncer Med 2018
MSK-IMPACT Yes
Hybrid- capture- based NGS
468 1,22 Mb NO MUT/Mb Ri zvi H JCO 2018
TruSight Tumor
170 - ILLUMINA No
Hybrid- capture- based NGS
170 0,524 Mb NO MUT/Mb In silico a na l ys es
Oncomine™
Tumor Mutation Load Assay - ThermoFisher
Scientific
No Amplicon-
based NGS 409 1,7 Mb NO MUT/Mb In silico a na l ys es
Issue: Harmonization
TMB_IEO experience: preliminary data
Panel
Diagnostic- Use/FDA approved
Chemistry # Genes Megabases Threshold Result Study
FoundationOne
CDx - Roche Yes
Hybrid- capture- based NGS
324 1,2 Mb YES
HIGH= ≥ 20 MUT/Mb INTERMEDIATE = 6-19 MUT/Mb
LOW = 1-5 MUT/Mb
Goodma n AM Mol Ca nc Ther 2017 Hel l ma nn MD NEJM 2018
CGP+ - Agilent SureSelect XT - Caris Molecular Intelligence
No
Hybrid- capture- based NGS
592 1,4 Mb YES HIGH= ≥ 17 MUT/Mb LOW = < 17 MUT/Mb
Ga tal i ca Z Eur Journa l Ca nc 2018 Va nderva l de A Ca ncer Med 2018
MSK-IMPACT Yes
Hybrid- capture- based NGS
468 1,22 Mb NO MUT/Mb Ri zvi H JCO 2018
TruSight Tumor
170 - ILLUMINA No
Hybrid- capture- based NGS
170 0,524 Mb NO MUT/Mb In silico a na l ys es
Oncomine™
Tumor Mutation Load Assay - ThermoFisher
Scientific
No Amplicon-
based NGS 409 1,7 Mb NO MUT/Mb In silico a na l ys es
TMB Assay & Samples
Panel
Diagnostic- Use/FDA approved
Chemistry # Genes Megabases Threshold Result Study
FoundationOne
CDx - Roche Yes
Hybrid- capture- based NGS
324 1,2 Mb YES
HIGH= ≥ 20 MUT/Mb INTERMEDIATE = 6-19 MUT/Mb
LOW = 1-5 MUT/Mb
Goodma n AM Mol Ca nc Ther 2017 Hel l ma nn MD NEJM 2018
CGP+ - Agilent SureSelect XT - Caris Molecular Intelligence
No
Hybrid- capture- based NGS
592 1,4 Mb YES HIGH= ≥ 17 MUT/Mb LOW = < 17 MUT/Mb
Ga tal i ca Z Eur Journa l Ca nc 2018 Va nderva l de A Ca ncer Med 2018
MSK-IMPACT Yes
Hybrid- capture- based NGS
468 1,22 Mb NO MUT/Mb Ri zvi H JCO 2018
TruSight Tumor
170 - ILLUMINA No
Hybrid- capture- based NGS
170 0,524 Mb NO MUT/Mb In silico a na l ys es
Oncomine™
Tumor Mutation Load Assay - ThermoFisher
Scientific
No Amplicon-
based NGS 409 1,7 Mb NO MUT/Mb In silico a na l ys es
RUO
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/Mb MSI-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 Marzo Elena Guerini-Rocco
Paolo Lopedote Alberto Ranghiero
Alessandra Rappa Mila Schiavi Francesco Spinelli
Tania Tamagni Davide Vacirca
Thank You
Molecular Diagnostics Unit
Division 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 te descritto rispetto agli altri due
* Maggiore correlazione con la risposta alla terapia 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
Chemistry # Genes Megabases Threshold Result Study
FoundationOne
CDx - Roche Yes
Hybrid- capture- based NGS
324 1,2 Mb YES
HIGH= ≥ 20 MUT/Mb INTERMEDIATE = 6-19 MUT/Mb
LOW = 1-5 MUT/Mb
Goodma n AM Mol Ca nc Ther 2017 Hel l ma nn MD NEJM 2018
CGP+ - Agilent SureSelect XT - Caris Molecular Intelligence
No
Hybrid- capture- based NGS
592 1,4 Mb YES HIGH= ≥ 17 MUT/Mb LOW = < 17 MUT/Mb
Ga tal i ca Z Eur Journa l Ca nc 2018 Va nderva l de A Ca ncer Med 2018
MSK-IMPACT Yes
Hybrid- capture- based NGS
468 1,22 Mb NO MUT/Mb Ri zvi H JCO 2018
TruSight Tumor
170 - ILLUMINA No
Hybrid- capture- based NGS
170 0,524 Mb NO MUT/Mb In silico a na l ys es
Oncomine™
Tumor Mutation Load Assay - ThermoFisher
Scientific
No Amplicon-
based NGS 409 1,7 Mb NO MUT/Mb In silico a na l ys es
