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Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells
Published on: February 8, 2018
Pan-cancer analysis of CD274 (PD-L1) mutations in 314,631 patient samples and subset correlation with PD-L1 protein
Richard S P Huang1, Brennan Decker2, Karthikeyan Murugesan2
1Foundation Medicine Inc, Cambridge, Massachusetts, USA rhuang@foundationmedicine.com.
Background:
The effects of non-amplification short variant (SV) mutations in CD274 (programmed death-ligand 1 (PD-L1)) on PD-L1 protein expression and immune checkpoint inhibitors (ICPIs) therapy are unknown. Here, we present a retrospective analysis of CD274 mutations detected by comprehensive genomic profiling (CGP) and correlate these results with tumor-cell PD-L1 immunohistochemistry (IHC)-based expression assessment to better understand the relationship between mutations and protein expression of PD-L1.
Methods:
CGP was performed on hybridization-captured, adaptor ligation-based libraries using DNA and/or RNA extracted from 314,631 tumor samples that were sequenced for up to 406 cancer-related genes and select gene rearrangements. PD-L1 IHC was performed on a subset of cases (n=58,341) using the DAKO 22C3 PD-L1 IHC assay and scored with the tumor proportion score (TPS).
Results:
Overall, the prevalence of CD274 SV mutations was low (0.3%, 1081/314,631) with 577 unique variants. The most common CD274 SV mutations were R260H (n=51), R260C (n=18), R125Q (n=12), C272fs*13 (n=11), R86W (n=10), and R113H (n=10). The prevalence of CD274 mutations varied depending on tumor type with diffuse large B-cell lymphoma (1.9%, 19/997), cutaneous squamous cell carcinoma (1.6%, 14/868), endometrial adenocarcinoma (1.0%, 36/3740), unknown primary melanoma (0.9%, 33/3679), and cutaneous melanoma (0.8%, 32/3874) having the highest frequency of mutations. Of the R260H cases concurrently tested with PD-L1 IHC, most (81.8%, 9/11) had no PD-L1 expression, which contrasts to the five E237K cases where most (80%, 4/5) had PD-L1 expression. In addition, we saw a significantly lower level of PD-L1 expression in samples with a clonal truncating variant (nonsense or frameshift indel) when compared with samples with a subclonal truncating variants (mean: TPS=1 vs TPS=38; p<0.001), and also in clonal versus subclonal missense mutations (mean: TPS=11 vs TPS=22, respectively; p=0.049) CONCLUSIONS: We defined the landscape of CD274 mutations in a large cohort of tumor types that can be used as a reference for examining CD274 mutations as potential resistance biomarkers for ICPI. Furthermore, we presented novel data on the correlation of CD274 mutations and PD-L1 protein expression, providing important new information on the potential functionality of these mutations and can serve as a basis for future research.
Insights
Non-amplification short variant mutations in programmed death-ligand 1 (PD-L1) are rare but impact PD-L1 protein expression. This study correlates CD274 mutations with PD-L1 levels, offering insights into immune checkpoint inhibitor therapy resistance.
Area of Science:
- Genomics
- Oncology
- Immunology
Background:
- The impact of non-amplification short variant (SV) mutations in CD274 (programmed death-ligand 1, PD-L1) on PD-L1 protein expression and immune checkpoint inhibitor (ICPI) therapy remains unclear.
- Understanding this relationship is crucial for predicting treatment response and resistance.
Purpose of the Study:
- To analyze the landscape of CD274 SV mutations across a large cohort of tumor types.
- To correlate CD274 mutations with PD-L1 protein expression assessed by immunohistochemistry (IHC).
- To investigate the potential of CD274 mutations as biomarkers for ICPI resistance.
Main Methods:
- Retrospective analysis of 314,631 tumor samples using comprehensive genomic profiling (CGP) to detect CD274 mutations.
- PD-L1 IHC assessment (DAKO 22C3 assay, tumor proportion score - TPS) on 58,341 samples.
- Correlation of identified CD274 mutations with PD-L1 expression levels.
Main Results:
- CD274 SV mutations were found in 0.3% of samples, with 577 unique variants identified.
- Prevalence varied by tumor type, notably higher in diffuse large B-cell lymphoma and cutaneous squamous cell carcinoma.
- Specific mutations like R260H were associated with absent PD-L1 expression, while E237K was linked to expression. Clonal truncating variants significantly reduced PD-L1 levels compared to subclonal variants.
Conclusions:
- Defined the landscape of CD274 mutations, providing a reference for their role as potential ICPI resistance biomarkers.
- Presented novel data correlating CD274 mutations with PD-L1 protein expression, highlighting the functional implications of these mutations.
- These findings form a basis for future research into CD274 mutations and ICPI therapy response.
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