Tingting Jiang1, Weiwei Shi1, W Fraser Symmans2, Charles Li1, James ... New Haven, CT 2University of Texas M D Anderson Cancer Center, Houston TX.
Broad Exonic DNA Diversity Is Associated with Resistance to Taxane-FAC Chemotherapy in Triple Negative Breast Cancer Tingting
1 Jiang ,
Weiwei
1Yale
1 Shi ,
W Fraser
2 Symmans ,
Charles
1 Li ,
James
1 Platt ,
Rosanna
2 Lau ,
Vikram B
1 Wali ,
Richard
1 Lifton ,
1 Pusztai ,
Lajos
Christos
1 Hatzis
Cancer Center, Yale School of Medicine, Yale University, New Haven, CT 2University of Texas M D Anderson Cancer Center, Houston TX Resistance is Associated with Broad Genomic Diversity
Gene
Results
Length (bp)
Pvalue
pCR
BIOCARTA_KERATINOCYTE_PATHWAY
0.022
pCR
AKAP95 role in mitosis and chromosome dynamics
0.028
pCR
Activation of camp-dependent protein kinase PKA
0.028
pCR
BIOCARTA_41BB_PATHWAY
0.028
pCR
BIOCARTA_STRESS_PATHWAY
0.028
pCR
Signaling events mediated by TCPTP
0.029
RD
Sonic hedgehog receptor PTC1 regulates cell cycle
0.043
pCR
PAR4-mediated thrombin signaling events
0.043
RD
BIOCARTA_CBL_PATHWAY
0.043
pCR
BIOCARTA_NKCELLS_PATHWAY
0.045
pCR
BIOCARTA_TFF_PATHWAY
0.045
RD
Higher in Group
MUC21
1701
5*10-4
RD
SLCO5A1
2547
0.027
pCR
We investigated the association of mutations in pathways with chemotherapy response in 677 pathways collected from NCI’s Pathway Interaction Database and from Biocarta. Of these, 14 pathways were identified as being significantly associated with response by Fisher exact test (Table 4).
We calculated overall mutational load and normal-adjusted clonal entropy of putative driver mutations as measures of broad genome heterogeneity.
0.04
pCR pCR
0.6
600
0.5
500
0.4
0.3
0.2
TOTAL_READS 250
Table 1. Somatic Mutations In Response Groups
RD pCR
0
Sample ID
192 (125-293)
eRD
59 (43-78)
223 (113-388)
0.008 0.006 Density
Density
0.004
4 3 2
150
1
SYNE1
SYNE1
0.002
63 (49-82)
SLCO5A1
MUC21
MUC21
Sample ID
100
200 0.1
0.2
0.3
0.4
0.5
0.6
400
N = 17
Figure 3. Hierarchical clustering of mutation patterns50 defines two response groups. Gene-level mutations (red) and wild type (yellow). On top of the heatmap, each sample is color coded for 0 response: eRD (red) or pCR (blue)
Bandwidth = 0.03631
600
800
0.7
N = 17
Clonal Entropy
●
350
● 0.20
0.25
0.30
0.35
ClonalEntropy entropy Clonal
Mutational Spectra of Chemo-resistant Tumors Differ All non-silent somatic mutations were combined in 6 transition/transversion patterns by different response groups. The mutational spectra were significantly different between pCR and eRD by Chi-square test (P=0.03). The proportion of C>T transition was higher in eRD tumors (P=0.011), whereas the A>G transition was lower (P=0.028) than in pCR. The same mutational spectrum shift was previously described in the comparison of trunk and branch driver events suggesting that eRD tumors may undergo heterogeneous branched evolution. 100% 90% 80%
P=0.011
70% 60% 50% 40%
Figure 6. Mutational spectra in different response groups. The difference between the spectra for driver mutations in pCR and eRD was assessed by Chi-square test. For specific type of mutation, two-tailed Fisher exact test was used to evaluate significance.
P=0.028
RD
P=0.03
Conclusions
RD pCR
RD pCR
0.000
pCR
Sample ID
Mutation load (all novel mutation)
SLCO5A1
●
●
pCR
Clonal heterogeneity
0
Response Group Non-silent in COSMIC Non-silent Novel Mean (range) Mean (range)
● ●
●
0%
0.0
200
●
10%
200
100
LRBA
● ●
●
●
300
0.1
LRBA
● ●
●● ● ●●
20%
400
612 M571 692 557 524 125 229 M635 696 485 M750 324 757 M792 402 665 658 661 494 735 M345 681 805 732 406 367 219 M211 348
Chemotherapy Response
●
●
30%
612 557 219 485 402 125 406 M211 692 494 696 M792 M750 805 348 735 665 732 M571 M635 524 661 M345 681 658 324 367 757 229
26394
0.04
RD pCR
Mutational Load
STNE1
8592
700 RD pCR
Clonal Entropy
LRBA
Mutational Load (Novel Mutations)
Mutation load (all novel mutation)
0.7
●
● ●●
Broad Genomic Variation As Marker of Response
Clonal Entropy Clonal heterogeneity
The mean coverage was over 150x and >90% of target regions had 20x data. We validated the specificity (98.2%) and sensitivity (86.7%) of the pipeline on the GIAB reference data.
300
0.021
Figure 5. Combination of broad genomic diversity measures for 29 TNBC tumors. Resistant tumors (red) tend to be associated with higher clonal entropy or higher novel mutational load.
●
250
pCR
200
0.009
Density
IIA IIIB IIIB IIB IIB IIIB IIB IIB IIB IIIA IIB IIIC IIIA IIIA IIA IIIB IIIA IIIA IIIA IIA IIIB IIIB IIIA IIB IIIA IIIB IIIB
GATA3 participate in activating the th2 cytokine genes expression BIOCARTA_EIF_PATHWAY
Density
N1 N3 N3 N1 N1 N3 N1 N1 N1 N2 N1 N3 N2 N2 N1 N3 N1 N1 N2 N0 N2 N2 N2 N0 N2 N3 N2
RD
Bandwidth = 20.1
Mutational Load
Figure 4. Measures of genomic variation on two response groups. Higher clonal entropy and higher mutational load appears to be associated with the resistant group (RD; red)
696 M211 402 125 M571 M792 524 681 M345 757 612 485 692 665 406 732 557 805 658 324 735 367 348 M635 229 494 219 661 M750
T2 T2 T3 T2 T2 T2 T2 T2 T2 T3 T2 T1 T2 T3 T1 T4 T3 T3 T2 T2 T4 T4 T2 T3 T2 T2 T4
TOTAL_READS (in Million)
3 3 3 3 3 2 3 3 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 3
M211 494 696 524 658 M750 665 661 612 557 485 367 125 348 757 732 681 406 219 229 M635 M345 402 805 M571 M792 735 324 692
pCR pCR pCR pCR pCR pCR pCR pCR pCR pCR pCR pCR pCR pCR RD RD RD RD RD RD RD RD RD RD RD RD RD
0.007
P53_tetramer
P53
Gene
524 557 658 661 665 692 696 735 805 M211 M571 M635 M750 M792 125 219 229 348 406 494 570 572 612 681 732 757 M345
internal ribosome entry pathway
P53_tetramer
P53
P53_TAD
Higher in Group
Proportion
E153_E154delinsX
D149E
R81X
G69fs
39_40del
R43H
P53_TAD
N107_S108delinsX
Exome Sequencing Analysis: TP53-pCR group • Raw reads filtered, trimmed and aligned to hg19 100 200 300 393 aa reference genome by Burrows-Wheeler Aligner mem.0 Patients and Samples • PCR duplicates removed by Picard and single TP53-eRD group nucleotide variants and indels were detected by Twenty nine TNBC samples were selected from a HaplotypeCaller in GATK. prospectively collected tissue bank of fine needle 0 100 200 300 393 aa aspiration (FNA) specimens obtained before preoperative • Low mapability and low concordance regions provided by Genome-In-A-Bottle (GIAB) were removed. Figure 2. Putative somatic mutations in TP53 in the two response chemotherapy to represent two extreme response • High quality variants annotated by ANNOVAR. groups. cohorts of pathologic complete response (pCR, N=18) and extensive residual cancer (eRD, N=11). No matched normal samples were available for this study. Germline Variant Filtering and Driver Mutations: Genomic Aberrations Associated with Response • External normal cohorts: 1000 genome project and Table 1. Clinical Information of 29 Sequenced 501 normal samples from TCGA. By aggregating mutations at the gene and pathway level, we identified Samples • Potential somatic mutations defined as :1) not present 4 genes (Table 3) and 14 pathways (Table 4) that were significantly Sample ID Response Grade Tumor Nodal AJCC in any of the 2 reference normal cohorts at frequency associated with response group. Group Stage Stage Stage > 0.01; or if 2) not present in > 6 of 29 tumors. 324 pCR 3 T2 N1 IIB Table 3. Mutated Genes Associated with Response • Putative driver mutations were then defined as non367 pCR 3 T4 N1 IIIB (Mutations aggregated at gene level; Fisher Exact Test) 402 pCR 3 T4 N2 IIIB synonymous mutations in 435 previously reported 485 pCR 3 T4 N2 IIIB cancer related genes (Pan-Cancer TCGA list).
Pvalue
150
NonSyn SNV Stopgain SNV Frameshift Subst
E153K
R141H
R141C
G113fs
C110S
N103fs
C85_T86delinsX
R81X
C44F
F80fs
NonSyn SNV Stopgain SNV Frameshift Subst
Pathway Name
●
RD pCR
● ●
Number of Mutation Load Novel nonsilent variant
Library preparation, Exome Capture and Sequencing: TP53 Most Frequently Mutated but Not Associated with Response • DNA extracted from samples in RNAlater and sheared to mean fragment length of ~140 bp TP53 TP53 - pCR Groupwas the most frequently mutated gene in 22 of 29 tumors, but • Exome capture by NimbleGen solution-capture exome had no significant association with response by the Fisher Exact array (SeqCap EZ version 2) test (P-value=0.67).
• 74-bp paired-end sequencing on Illumina HiSeq 2000 instrument at the Yale Center for Genome Analysis. TP53 - eRD Group R43H
Previous efforts to develop transcriptional markers of chemotherapy sensitivity in TNBC had limited success due to the heterogeneity of this disease. The purpose of this study was 1) to identify potential genomic differences between extremely chemotherapy sensitive and highly chemotherapy resistant TNBC through whole exome sequencing and 2) to assess whether broader measures of genomic heterogeneity maybe predictive of resistance to chemotherapy.
Table 4. Mutated Pathways Significantly Associated with Response (Fisher Exact test)
100
Genomic Aberrations Associated with Response
Whole Exome Sequencing
Introduction
• Response specific mutational patterns in 4 genes and 14 pathways were detected, but significance of association was rather weak. • Chemotherapy resistant TNBC have greater genomic diversity and show distinctive mutational spectra compared to highly chemotherapy sensitive tumors. • Our analysis suggests that broad measures of genomic diversity may serve as markers of resistance to chemotherapy.
