Title of the Poster Presentation Goes Here Authors of the Poster Presentation Goes Here Institutional and/or Graduate School of Biomedical Sciences Affiliation Goes Here Transcriptional profiling and consensus molecular subtype (CMS) assignment to understand response and resistance to anti-EGFR therapy in colorectal cancer Saikat Chowdhury 1, *, Ria Gupta 1 , Valsala Haridas 1 , Mohammad A. Zeineddine 1 , Scott Kopetz 1 , John Paul Shen 1 1 Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA * Presenting author: schowdhury4@mdanderson.org
Background • Activating mutations in the extended RAS ( KRAS, NRAS , and BRAF) genes confer resistance to anti-EGFR Ab (e.g., Cetuximab) therapy of colorectal cancer (CRC) patients 1 • Left sided RAS WT tumors are incorporated in NCCN guideline as predictor of anti-EGFR Ab therapy 2 • However, only 40% - 45% RAS WT patients have been found to respond to the therapy 3 • Identification of novel biomarkers is required for better stratification of the metastatic CRC patients for anti-EGFR Ab therapy Materials and Methods • Gene expression datasets of two retrospective clinical cohorts and two pre-clinical cohorts were downloaded from Gene Expressions Omnibus Objective • Identification of transcriptomic determinants for predicting and understanding the primary resistance mechanisms to anti- EGFR antibody therapy of colorectal cancer patients
Results • Percentages of clinically benefited CMS2 RAS WT patients (92% and 68%), PDX models (84%), and sensitive CRC cell lines (60%) were highest in both clinical and preclinical cohorts
• HT55 and SNUC1 cell lines were resistant to cetuximab (IC50 >10µg/ml) • Single agent inhibitors of E2F (AZD7762 and MK-8776), mTOR (Everolimus), and MYC (JQ1) pathways could not inhibit HT55 and SNUC1 cell growth (IC50 > 10µM) Pharmacological parameters of cell viability assays of HT55 and SNUC1 lines
• Response to anti-EGFR antibody therapy was higher for left- sided (DCR: ~83% (58/70) vs 62.5% (5/8) for right-sided) tumors • However, after controlling for CMS, DCR for right and left sided CMS2/ RAS WT tumors were similar
Cell viability assays
HT55
SNUC1
Single-drug assay
IC50
AUC IC50
AUC
Cetuximab (0.01*/0.1 $ µg/ml – 100*/50 $ µg/ml)
> 10 µg/ml
383 > 10 µg/ml
251
AZD7762 (0.8 µM – 10 µM) MK-8776 (1.25 µM – 20 µM)
4.9 µM 159
> 10 µM 180
> 10 µM 108 > 10 µM 111 > 10 µM 123 > 10 µM 136 > 10 µM 97.1 > 10 µM 90.7
JQ1 (0.25 µM – 10 µM)
Everolimus (1 µM – 15 µM)
*HT55 cell line assay; $ SNUC1 cell line assay
• The addition of AZD7762, MK-8776, Everolimus, and JQ1 significantly decreased HT55 cell viability relative to cetuximab alone
• MTORC1, E2F, and MYC pathway gene-sets were found significantly enriched (FDR < 0.3) in the cetuximab refractory CMS2 RAS WT samples of both clinical and preclinical cohorts
• Median PFS (7.47 months and 3.06 months) of CMS2 RAS WT patients in two clinical cohorts were higher than the other CMS subclasses b
Transcriptomic datasets used in this study
Total Samples
Sample types
Datasets
Treatment
References
Cell lines
CRC Cell lines
Medico et al., Nat Comm, 2015
GSE59857
146
Cetuximab
PDX Models
• However, combinatorial effect of these drugs with cetuximab was additive, rather than synergistic Conclusions • CMS may be used as biomarker for classifying RAS WT & MSS CRC patients for anti-EGFR antibody therapy • Both left and right-sided RAS WT /MSS/CMS2 tumors may be benefited from cetuximab therapy • RAS-MAPK independent signaling pathways may regulate primary resistance to anti-EGFR antibodies
Bertotti et al., Nature, 2015
GSE76402
216
PDX Models Cetuximab
Okita et al.
Patient Cohorts
Khambata-Ford et al.
FOLFOX/ FOLFIRI & Cetuximab
pCRC tissues pCRC tissues
Okita et al., Oncotarget,2018
• RAS gene mutation and CMS of CRC patients were significantly associated with PFS (p-value < 0.05) • CMS1 and CMS2 patients showed worst and best prognosis to anti-EGFR treatment, respectively
GSE104645
135
Khambata-Ford et al., J Clin Oncol, 2007
GSE5851
68
Cetuximab
• Workflow:
Clinical cohorts
Pre-clinical cohorts
Cox proportional hazard analysis of PFS of anti-EGFR treatment in clinical cohorts
• Single sample GSEA revealed that MTORC1, E2F, and MYC pathways were heterogeneously active (NES > 1) in the cetuximab refractory RAS WT samples
Okita et al. cohort
Khambata-Ford et al. cohort
Gene expressions
Univariate Study Multivariate Study HR P-value HR P-value HR P-value HR P-value Multivariate Study Univariate Study
Variables
References 1. Therkildsen C, et al., Acta Oncol, 2014;53(7):852-64 2. National Comprehensive Cancer Network. Colon Cancer (Version 1.2021) 3. Vidal J, et al., AnnOncol, 2019;30(3):439-46
RAS WT & MSS Samples
CMS2 RAS WT Non-responder patients
Age Sex
0.99
ns
0.99
0.63 0.99 0.94 1.00 0.78
CMSClassifier & CMSCaller
VIPER
Female vs Male
1.14
n
0.94
0.79 1.85 0.02*
1.62 0.11
RAS gene mutation
Protein Activity Scores
Consensus molecular subtypes (CMS)
9.44E- 07*
8.43E- 07*
1.00E- 02*
1.40E- 01
Funding
RAS wt vs RAS mut
0.34
0.31
0.49
0.61
Tumor sides
GSEA & ssGSEA
Acknowledgement This work was supported by the National Cancer Institute (L30 CA171000 and K22 CA234406 to J.P.S., and The Cancer Center Support Grant P30 CA016672), the Cancer Prevention & Research Institute of Texas (RR180035 to J.P.S., J.P.S. is a CPRIT Scholar in Cancer Research), and the Col. Daniel Connelly Memorial Fund. This study was also supported by the Colorectal Cancer Moonshot Program and SPORE program (P50CA221707) of The UT MD Anderson Cancer Center. We acknowledge the support of the High-Performance Computing for research facility at The University of Texas MD Anderson Cancer Center for providing computational resources that have contributed to the research results reported in this work.
Left vs right
0.68
0.11
0.89
0.66
NA NA NA NA
Cetuximab treatments vs CMS
Okita et al. cohort
Khambata-Ford et al. cohort
CMS CMS1 vs CMS2/3/4 2.19 0.0038* CMS2 vs CMS1/3/4 0.55 0.002*
3.47 0.002* 0.44 0.001*
7.10 0.0009* 0.44 0.003*
1.44 0.62
0.33 0.03 0.99 0.05
CMS2 RAS WT PDX models
CMS2 RAS WT CRC cell lines
(PFS)
Resistant pathways
(RECIST, AUC)
CMS3 vs CMS1/2/4 CMS4 vs CMS1/2/3
1.2
0.357 1.00
NA NA NA NA
1.22 0.06 1.59 0.09 • Age, sex, and primary tumor sidedness (right vs. left colon) were not significantly associated (p-value > 0.05) with progression-free survival (PFS) of cetuximab treatment 0.32 1.57 1.6
Drug screening
Clinical benefits & Sensitivities of CMS
Survival analyses of CMS
In vitro validation on HT55 and SNUC1 cell lines
Medico et al. cohort
Bertotti et al. cohort
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