1. Correlation Between Gene Expression and Prognostic Biomarkers in Small Cell Bladder Cancer (SCBC)
Vadim S Koshkin1, Andrew Dhawan1, Ming Hu1, Jordan Reynolds1, Paul Elson1, Jesse McKenney1, Laura R Saunders2, Kumiko Isse2, Summya Rashid1, Aysegul Balyimez1, Moshe C Ornstein1, Timothy D Gilligan1, Byron Lee1, Jacob G Scott1, Brian I Rini1, Jorge A Garcia1, Petros Grivas1,3, Omar Y Mian1
1. Cleveland Clinic, Cleveland, OH 2. AbbVie Stemcentrx, San Francisco, CA 3. University of Washington, Seattle, WA
Background
Figure 1: Analysis Schema
Results
Results
Small Cell Bladder Cancer (SCBC) is a rare histologic variant with a poorly understood biology whose treatment patterns are extrapolated from small cell lung cancer (SCLC) and urothelial carcinoma
DLL3 is a Notch pathway protein overexpressed in neuroendocrine malignancies and targeted by an antibody-drug conjugate (Rova-T) which has shown anti-tumor efficacy in early phase trials in SCLC1,2
In prior analyses we found that increased protein expression of DLL3 and CD56 as well as increased small cell % predict worse outcomes in SCBC3
The Cancer Genome Atlas (TCGA) data in bladder cancer suggests the presence of a neuronal subtype misclassified by conventional histopathology which can be identified based on molecular subtyping1
Association of gene expression data with available biomarkers can improve risk stratification and inform treatment decisions
Figure 3: Gene Expression of Relevant Protein Biomarkers in Tumor Relative to Normal
Table 5: Correlations of Small Cell Component With Relevant Genes (N=39) A.
Tumor vs Normal
Gene Expression
DLL3
NCAM1
DLL4
CHGA
DLL1
ENO1
EZH2
RB1
Pearson Correlation
0.383
0.418
0.308
-0.035
0.069
0.194
-0.251
HES1
HES5
HEY1
NOTCH1
NOTCH2
NOTCH4
SYP
TP53
-0.343
-0.077
0.207
-0.173
-0.195
-0.124
0.289
-0.004 B
DLL3 C
NCAM1/CD56
DLL3
NCAM1
Figure 2: Gene Expression Analysis in Normal and Tumor Tissue in 39 Patients
log-fold-change
Expression
Expression
A: Small Cell Bladder Cancer Tissue Origin
B: Gene Expression Heterogeneity by Site (PCA)
*Values in red are statistically significant at p<0.05
Conclusions
Tumor
Normal
Tumor
Normal
Average log CPM
(A) Gene expression log fold change vs average log of counts per million (CPM) across samples, significantly differentially expressed genes (log fold change >1.0, adj p<0.05) are displayed in red if upregulated and blue if downregulated in tumors vs normal. Arrows highlight genes of interest. Plots for (B) DLL3 and (C) NCAM1/CD56 display gene expression in individual tumor and normal samples.
Expression of genes implicated in the pathophysiology of small cell and neuroendocrine tumors correlated with biomarkers previously shown to be prognostic in SCBC: protein expression of DLL3 and CD56/NCAM1 and small cell component of tumor
Strong correlation between gene and protein expression of both DLL3 and NCAM1 suggest their regulation at the transcriptional level
Notable genes whose expression correlated with relevant biomarkers in this preliminary analysis include DLL3, NCAM1, DLL4, NOTCH1, CHGA, SYP
Using these and others as seed genes in a network-based approach, our aim is to develop a prognostic gene expression signature in SCBC, then validate this signature in external patient cohorts while also pursuing a comparison with bladder cancer TCGA and small cell lung cancer datasets
Tissue
Metastasis
Tumor
Normal
Table 2: Correlations of DLL3 Protein Expression With Relevant Genes (N=39)
A: Diagram of tissues of origin utilized in the gene expression analysis. Green represents normal urothelial tissue samples, yellow represents tissue samples from primary bladder tumor, and red represents tissue from metastatic sample.
B: Three-dimensional representation of the principal component analysis which represents tissue gene expression heterogeneity across 2560 genes as a Euclidian distance in three principal components. This displays a similarity in gene expression among normal urothelial samples (green), which stand apart from gene expression similarity observed across primary SCBC tumors (yellow).
Gene Expression
DLL3
NCAM1
DLL4
CHGA
DLL1
ENO1
EZH2
RB1
Pearson Correlation
0.699
0.113
0.448
0.549
0.050
-0.336
-0.291
-0.484
HES1
HES5
HEY1
NOTCH1
NOTCH2
NOTCH4
SYP
TP53
-0.116
0.071
0.085
-0.475
-0.075
-0.068
0.151
-0.252
Methods
Biomarkers of interest were identified based on differential gene expression analyses comparing tumor tissue samples and normal urothelium (Fig. 3)
Associations of protein expression of biomarkers of interest (DLL3 and CD56/NCAM1) as well as small cell % with the expression of genes relevant in pathophysiology of small cell tumors (Table 1) were analyzed to identify biomarkers pertinent to molecular diagnostics (Pearson correlation, p < 0.05)
*Values in red are statistically significant at p<0.05
63 SCBC patients seen at Cleveland Clinic had small cell % independently confirmed and quantified at the time of analysis by an experienced GU pathologist
52 patients had tumor tissue available for immunohistochemistry (IHC) of biomarkers of interest including DLL3 and CD56, while 39 of those patients had tissue available for gene expression profiling using HTG EdgeSeq Oncology Biomarker Panel (OBP) with probes for 2560 genes (Fig. 1)
39 patients assessed for gene expression had 46 total samples: 39 primary SCBC tumors, 6 adjacent normal urothelium and 1 metastatic sample (Fig. 2)
Table 3: Correlations of CD56/NCAM1 Protein Expression With Relevant Genes (N=39)
Gene Expression
DLL3
NCAM1
DLL4
CHGA
DLL1
ENO1
EZH2
RB1
Pearson Correlation
0.195
0.615
0.379
0.266
0.186
-0.022
0.040
-0.088
HES1
HES5
HEY1
NOTCH1
NOTCH2
NOTCH4
SYP
TP53
-0.068
-0.352
0.419
-0.340
-0.218
0.007
0.342
-0.213
*Values in red are statistically significant at p<0.05
References
Table 4: Protein Expression of Relevant Biomarkers (N=39)
Table 1: Panel of Genes Proposed for a Potential Signature
1. Saunders et al. Sci Transl Med, 2015
2. Rudin et al. Lancet Oncol, 2017
3. Koshkin et al. J Clin Oncol 36, 2018 Abstract 452
4. Robertson et al. Cell, 2017
Biomarker
CD56
DLL3
Small Cell % (≥50%)
% of Tumors with Expression
81%
72%
87%
Median (% of Cells with Expression)
65%
60%
100%
Range (% of Cells)
0-100%
0-95%
5-100%
DLL1
CHGA
HES1
NOTCH1
RB1
EZH2
DLL3
NCAM1
HES5
NOTCH2
SYP
DLL4
ENO1
HEY1
NOTCH4
TP53
Please send correspondence to: