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Whole-exome sequencing of pancreatic cancer defines genetic diversity and therapeutic targets.

Witkiewicz AK, McMillan EA, Balaji U, Baek G, Lin WC, Mansour J, Mollaee M, Wagner KU, Koduru P, Yopp A, Choti MA, Yeo CJ, McCue P, White MA, Knudsen ES - Nat Commun (2015)

Bottom Line: Copy number alterations target multiple tumour suppressive/oncogenic loci; however, amplification of MYC is uniquely associated with poor outcome and adenosquamous subtype.KRAS mutations are observed in >90% of cases, but codon Q61 alleles are selectively associated with improved survival.Together, these data delineate new genetic diversity of PDA and provide insights into prognostic determinants and therapeutic targets.

View Article: PubMed Central - PubMed

Affiliation: 1] Simmons Cancer Center, UT Southwestern Medical Center, Dallas, Texas 75390, USA [2] Department of Pathology, UT Southwestern Medical Center, Dallas, Texas 75390, USA.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDA) has a dismal prognosis and insights into both disease etiology and targeted intervention are needed. A total of 109 micro-dissected PDA cases were subjected to whole-exome sequencing. Microdissection enriches tumour cellularity and enhances mutation calling. Here we show that environmental stress and alterations in DNA repair genes associate with distinct mutation spectra. Copy number alterations target multiple tumour suppressive/oncogenic loci; however, amplification of MYC is uniquely associated with poor outcome and adenosquamous subtype. We identify multiple novel mutated genes in PDA, with select genes harbouring prognostic significance. RBM10 mutations associate with longer survival in spite of histological features of aggressive disease. KRAS mutations are observed in >90% of cases, but codon Q61 alleles are selectively associated with improved survival. Oncogenic BRAF mutations are mutually exclusive with KRAS and define sensitivity to vemurafenib in PDA models. High-frequency alterations in Wnt signalling, chromatin remodelling, Hedgehog signalling, DNA repair and cell cycle processes are observed. Together, these data delineate new genetic diversity of PDA and provide insights into prognostic determinants and therapeutic targets.

No MeSH data available.


Related in: MedlinePlus

Copy number alterations in PDA.(a) APC clustering of PDA cases by CNV revealed several distinct clades and associated clusters. Clusters 5 and 6 exhibit higher overall CNV relative to the other clusters. (b) Clusters 5 and 6 are enriched for mutations or homozygous deletion of genes involved in double-strand break repair, but not in TP53. Significance was determined by hypergeometric test. (c) Cluster 5 and 6 trend towards poor survival relative to clusters with less CNV. Hazard ratio and P values were obtained from Cox proportional hazard test. (d) GISTIC analysis of cases reveals chromosomal regions that are significantly deleted/amplified. (e) Kaplan–Meier analysis of the association of the 8q24.13 locus amplification with overall survival. P value was obtained from Cox proportional hazard test. (f) Fluorescence in situ hybridization with MYC break apart probes demonstrates amplification in the absence of translocation at the MYC locus (Scale bar, 5 μM).
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f2: Copy number alterations in PDA.(a) APC clustering of PDA cases by CNV revealed several distinct clades and associated clusters. Clusters 5 and 6 exhibit higher overall CNV relative to the other clusters. (b) Clusters 5 and 6 are enriched for mutations or homozygous deletion of genes involved in double-strand break repair, but not in TP53. Significance was determined by hypergeometric test. (c) Cluster 5 and 6 trend towards poor survival relative to clusters with less CNV. Hazard ratio and P values were obtained from Cox proportional hazard test. (d) GISTIC analysis of cases reveals chromosomal regions that are significantly deleted/amplified. (e) Kaplan–Meier analysis of the association of the 8q24.13 locus amplification with overall survival. P value was obtained from Cox proportional hazard test. (f) Fluorescence in situ hybridization with MYC break apart probes demonstrates amplification in the absence of translocation at the MYC locus (Scale bar, 5 μM).

Mentions: Affinity propagation clustering (APC) was used to delineate deterministic patterns of commonality associated with copy number alterations (Fig. 2a, left panel) and partitioned cases in discrete subtypes with increasing genetic complexity (Fig. 2a, right panel). Cases with high levels of amplifications/deletions (clusters 5 and 6) were significantly over-represented for alterations of genes involved in DNA break repair, but not TP53 (Fig. 2b). Similar results were observed with standard hierarchical clustering methods based on Euclidean distance (Supplementary Figs 12 and 13). Both cluster 5 and 6 harboured poor outcome relative to other clusters with fewer copy number changes (Fig. 2c, Supplementary Figs 12 and 13). GISTIC analysis defined significant common regions of amplification and deletion that harbour multiple oncogenes (for example, MYC and CCND1) and tumour suppressors (for example, SMAD4 and CDKN2A) (Fig. 2d and Supplementary Data 2). Interrogation of amplified and deleted regions for association with survival (Supplementary Data 2) revealed that the amplification of the 8q24 locus, harbouring the MYC oncogene, was uniquely associated with poor outcome (Fig. 2e, Supplementary Data 2). While MYC overexpression has been shown to facilitate the development of pancreatic cancer in mouse models15, little analysis has been performed in patient specimens. The amplification of MYC was confirmed by fluorescent in situ hybridization (Fig. 2f). The MYC amplified cases did not have a higher mutation burden or association with other hallmark mutations of PDA; however, amplification was significantly over-represented in the adenosquamous subtype of pancreatic carcinoma (Supplementary Fig. 14). Evaluation of precursor lesions pancreatic intraepithelial neoplasia (PanIN) associated with invasive disease also revealed MYC amplification (Supplementary Fig. 15). These data suggest a role of MYC in initiation and progression of this exceedingly aggressive form of PDA. Consistent with this observation, in a MYC-driven mouse model of pancreatic cancer, the tumours exhibited adenosquamous histology and stained positively for p63, an established marker of squamous differentiation (Supplementary Fig. 16).


Whole-exome sequencing of pancreatic cancer defines genetic diversity and therapeutic targets.

Witkiewicz AK, McMillan EA, Balaji U, Baek G, Lin WC, Mansour J, Mollaee M, Wagner KU, Koduru P, Yopp A, Choti MA, Yeo CJ, McCue P, White MA, Knudsen ES - Nat Commun (2015)

Copy number alterations in PDA.(a) APC clustering of PDA cases by CNV revealed several distinct clades and associated clusters. Clusters 5 and 6 exhibit higher overall CNV relative to the other clusters. (b) Clusters 5 and 6 are enriched for mutations or homozygous deletion of genes involved in double-strand break repair, but not in TP53. Significance was determined by hypergeometric test. (c) Cluster 5 and 6 trend towards poor survival relative to clusters with less CNV. Hazard ratio and P values were obtained from Cox proportional hazard test. (d) GISTIC analysis of cases reveals chromosomal regions that are significantly deleted/amplified. (e) Kaplan–Meier analysis of the association of the 8q24.13 locus amplification with overall survival. P value was obtained from Cox proportional hazard test. (f) Fluorescence in situ hybridization with MYC break apart probes demonstrates amplification in the absence of translocation at the MYC locus (Scale bar, 5 μM).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4403382&req=5

f2: Copy number alterations in PDA.(a) APC clustering of PDA cases by CNV revealed several distinct clades and associated clusters. Clusters 5 and 6 exhibit higher overall CNV relative to the other clusters. (b) Clusters 5 and 6 are enriched for mutations or homozygous deletion of genes involved in double-strand break repair, but not in TP53. Significance was determined by hypergeometric test. (c) Cluster 5 and 6 trend towards poor survival relative to clusters with less CNV. Hazard ratio and P values were obtained from Cox proportional hazard test. (d) GISTIC analysis of cases reveals chromosomal regions that are significantly deleted/amplified. (e) Kaplan–Meier analysis of the association of the 8q24.13 locus amplification with overall survival. P value was obtained from Cox proportional hazard test. (f) Fluorescence in situ hybridization with MYC break apart probes demonstrates amplification in the absence of translocation at the MYC locus (Scale bar, 5 μM).
Mentions: Affinity propagation clustering (APC) was used to delineate deterministic patterns of commonality associated with copy number alterations (Fig. 2a, left panel) and partitioned cases in discrete subtypes with increasing genetic complexity (Fig. 2a, right panel). Cases with high levels of amplifications/deletions (clusters 5 and 6) were significantly over-represented for alterations of genes involved in DNA break repair, but not TP53 (Fig. 2b). Similar results were observed with standard hierarchical clustering methods based on Euclidean distance (Supplementary Figs 12 and 13). Both cluster 5 and 6 harboured poor outcome relative to other clusters with fewer copy number changes (Fig. 2c, Supplementary Figs 12 and 13). GISTIC analysis defined significant common regions of amplification and deletion that harbour multiple oncogenes (for example, MYC and CCND1) and tumour suppressors (for example, SMAD4 and CDKN2A) (Fig. 2d and Supplementary Data 2). Interrogation of amplified and deleted regions for association with survival (Supplementary Data 2) revealed that the amplification of the 8q24 locus, harbouring the MYC oncogene, was uniquely associated with poor outcome (Fig. 2e, Supplementary Data 2). While MYC overexpression has been shown to facilitate the development of pancreatic cancer in mouse models15, little analysis has been performed in patient specimens. The amplification of MYC was confirmed by fluorescent in situ hybridization (Fig. 2f). The MYC amplified cases did not have a higher mutation burden or association with other hallmark mutations of PDA; however, amplification was significantly over-represented in the adenosquamous subtype of pancreatic carcinoma (Supplementary Fig. 14). Evaluation of precursor lesions pancreatic intraepithelial neoplasia (PanIN) associated with invasive disease also revealed MYC amplification (Supplementary Fig. 15). These data suggest a role of MYC in initiation and progression of this exceedingly aggressive form of PDA. Consistent with this observation, in a MYC-driven mouse model of pancreatic cancer, the tumours exhibited adenosquamous histology and stained positively for p63, an established marker of squamous differentiation (Supplementary Fig. 16).

Bottom Line: Copy number alterations target multiple tumour suppressive/oncogenic loci; however, amplification of MYC is uniquely associated with poor outcome and adenosquamous subtype.KRAS mutations are observed in >90% of cases, but codon Q61 alleles are selectively associated with improved survival.Together, these data delineate new genetic diversity of PDA and provide insights into prognostic determinants and therapeutic targets.

View Article: PubMed Central - PubMed

Affiliation: 1] Simmons Cancer Center, UT Southwestern Medical Center, Dallas, Texas 75390, USA [2] Department of Pathology, UT Southwestern Medical Center, Dallas, Texas 75390, USA.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDA) has a dismal prognosis and insights into both disease etiology and targeted intervention are needed. A total of 109 micro-dissected PDA cases were subjected to whole-exome sequencing. Microdissection enriches tumour cellularity and enhances mutation calling. Here we show that environmental stress and alterations in DNA repair genes associate with distinct mutation spectra. Copy number alterations target multiple tumour suppressive/oncogenic loci; however, amplification of MYC is uniquely associated with poor outcome and adenosquamous subtype. We identify multiple novel mutated genes in PDA, with select genes harbouring prognostic significance. RBM10 mutations associate with longer survival in spite of histological features of aggressive disease. KRAS mutations are observed in >90% of cases, but codon Q61 alleles are selectively associated with improved survival. Oncogenic BRAF mutations are mutually exclusive with KRAS and define sensitivity to vemurafenib in PDA models. High-frequency alterations in Wnt signalling, chromatin remodelling, Hedgehog signalling, DNA repair and cell cycle processes are observed. Together, these data delineate new genetic diversity of PDA and provide insights into prognostic determinants and therapeutic targets.

No MeSH data available.


Related in: MedlinePlus