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Intra-tumor genetic heterogeneity and alternative driver genetic alterations in breast cancers with heterogeneous HER2 gene amplification.

Ng CK, Martelotto LG, Gauthier A, Wen HC, Piscuoglio S, Lim RS, Cowell CF, Wilkerson PM, Wai P, Rodrigues DN, Arnould L, Geyer FC, Bromberg SE, Lacroix-Triki M, Penault-Llorca F, Giard S, Sastre-Garau X, Natrajan R, Norton L, Cottu PH, Weigelt B, Vincent-Salomon A, Reis-Filho JS - Genome Biol. (2015)

Bottom Line: HER2 is overexpressed and amplified in approximately 15% of invasive breast cancers, and is the molecular target and predictive marker of response to anti-HER2 agents.In a subset of these cases, heterogeneous distribution of HER2 gene amplification can be found, which creates clinically challenging scenarios.Our results indicate that even driver genetic alterations, such as HER2 gene amplification, can be heterogeneously distributed within a cancer, and that the HER2-negative components are likely driven by genetic alterations not present in the HER2-positive components, including BRF2 and DSN1 amplification and HER2 somatic mutations.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA. ngk1@mskcc.org.

ABSTRACT

Background: HER2 is overexpressed and amplified in approximately 15% of invasive breast cancers, and is the molecular target and predictive marker of response to anti-HER2 agents. In a subset of these cases, heterogeneous distribution of HER2 gene amplification can be found, which creates clinically challenging scenarios. Currently, breast cancers with HER2 amplification/overexpression in just over 10% of cancer cells are considered HER2-positive for clinical purposes; however, it is unclear as to whether the HER2-negative components of such tumors would be driven by distinct genetic alterations. Here we sought to characterize the pathologic and genetic features of the HER2-positive and HER2-negative components of breast cancers with heterogeneous HER2 gene amplification and to define the repertoire of potential driver genetic alterations in the HER2-negative components of these cases.

Results: We separately analyzed the HER2-negative and HER2-positive components of 12 HER2 heterogeneous breast cancers using gene copy number profiling and massively parallel sequencing, and identified potential driver genetic alterations restricted to the HER2-negative cells in each case. In vitro experiments provided functional evidence to suggest that BRF2 and DSN1 overexpression/amplification, and the HER2 I767M mutation may be alterations that compensate for the lack of HER2 amplification in the HER2-negative components of HER2 heterogeneous breast cancers.

Conclusions: Our results indicate that even driver genetic alterations, such as HER2 gene amplification, can be heterogeneously distributed within a cancer, and that the HER2-negative components are likely driven by genetic alterations not present in the HER2-positive components, including BRF2 and DSN1 amplification and HER2 somatic mutations.

No MeSH data available.


Related in: MedlinePlus

Sequencing analysis of HER2-positive and HER2-negative components of HER2 heterogeneous breast cancers identified founder genetic events and intra-tumor mutational heterogeneity. (A) Clonal frequencies of mutations identified in HER2-positive and HER2-negative components of HER2 heterogeneous breast cancers T6, T11 and T12, which were subjected to whole exome sequencing and orthogonal validation by amplicon sequencing (Ion Torrent) or targeted capture massively parallel sequencing (Illumina). Clonal mutation frequencies were estimated from the mutant allelic fractions adjusted according to tumor cellularity, tumor ploidy and local copy number states using ABSOLUTE [47]. Indel, insertion and deletion; SNV, single nucleotide variant. (B) Diagram illustrating the cancer cell fraction, as defined by ABSOLUTE, of mutations identified in cases T6, T11 and T12. Note the presence of subclonal mutations in the HER2-negative components of all cases, and in the HER2-positive components of cases T6 and T12. (C) Allelic fractions of mutations identified in HER2-positive and HER2-negative components of HER2 heterogeneous breast cancers obtained through targeted massively parallel sequencing analysis using a panel of 273 genes comprising genes frequently mutated in breast cancer and DNA repair-related genes. Indel, insertion and deletion; SNV, single nucleotide variant.
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Fig5: Sequencing analysis of HER2-positive and HER2-negative components of HER2 heterogeneous breast cancers identified founder genetic events and intra-tumor mutational heterogeneity. (A) Clonal frequencies of mutations identified in HER2-positive and HER2-negative components of HER2 heterogeneous breast cancers T6, T11 and T12, which were subjected to whole exome sequencing and orthogonal validation by amplicon sequencing (Ion Torrent) or targeted capture massively parallel sequencing (Illumina). Clonal mutation frequencies were estimated from the mutant allelic fractions adjusted according to tumor cellularity, tumor ploidy and local copy number states using ABSOLUTE [47]. Indel, insertion and deletion; SNV, single nucleotide variant. (B) Diagram illustrating the cancer cell fraction, as defined by ABSOLUTE, of mutations identified in cases T6, T11 and T12. Note the presence of subclonal mutations in the HER2-negative components of all cases, and in the HER2-positive components of cases T6 and T12. (C) Allelic fractions of mutations identified in HER2-positive and HER2-negative components of HER2 heterogeneous breast cancers obtained through targeted massively parallel sequencing analysis using a panel of 273 genes comprising genes frequently mutated in breast cancer and DNA repair-related genes. Indel, insertion and deletion; SNV, single nucleotide variant.

Mentions: To determine if the constellations of mutations would be distinct between the HER2-positive and HER2-negative components of HER2 heterogeneous breast cancers, and to identify potential driver mutations restricted to the HER2-negative components, we subjected the HER2-positive and HER2-negative components of three cases (that is, T6, T11 and T12), for which sufficient DNA from frozen tumor and matched normal tissues was available, to whole exome sequencing. Selected somatic mutations identified were validated by high-depth amplicon sequencing (Ion Torrent, 4000×) or targeted massively parallel sequencing (Figure 5A; Additional files 8, 9 and 10). Analysis of the clonal frequencies using ABSOLUTE [47] revealed that known founder genetic events such as somatic mutations in TP53 and/or PIK3CA were shared and inferred to be present in all cells of both the HER2-positive and HER2-negative components of these cases (Table 1; Figure 5A). This analysis also revealed the presence of subclonal mutations in the HER2-negative components of all cases, and in the HER2-positive component of cases T6 and T12 (Figure 5A,B). We next performed targeted massively parallel sequencing, using a panel of 273 genes frequently mutated in breast cancer and DNA repair-related genes [48], of the HER2-negative and HER2-positive components of 5 HER2 heterogeneous breast cancers (that is, T1, T3, T4, T8, and T9; Figure 5C). Consistent with the observations made by whole exome sequencing analysis, in all cases, the HER2-negative and HER2-positive components harbored somatic mutations in common, including TP53 somatic mutations in three cases (Figure 5C). Interestingly, in the two TP53 wild-type cases subjected to targeted sequencing, we identified an ARID1A mutation (that is, T4) and PIK3CA and CBFB mutations (that is, T8), which were common to the two components (Figure 5C). Taken together, in all cases analyzed, the HER2-negative and HER2-positive components shared identical somatic mutations, indicating their clonal relatedness.Figure 5


Intra-tumor genetic heterogeneity and alternative driver genetic alterations in breast cancers with heterogeneous HER2 gene amplification.

Ng CK, Martelotto LG, Gauthier A, Wen HC, Piscuoglio S, Lim RS, Cowell CF, Wilkerson PM, Wai P, Rodrigues DN, Arnould L, Geyer FC, Bromberg SE, Lacroix-Triki M, Penault-Llorca F, Giard S, Sastre-Garau X, Natrajan R, Norton L, Cottu PH, Weigelt B, Vincent-Salomon A, Reis-Filho JS - Genome Biol. (2015)

Sequencing analysis of HER2-positive and HER2-negative components of HER2 heterogeneous breast cancers identified founder genetic events and intra-tumor mutational heterogeneity. (A) Clonal frequencies of mutations identified in HER2-positive and HER2-negative components of HER2 heterogeneous breast cancers T6, T11 and T12, which were subjected to whole exome sequencing and orthogonal validation by amplicon sequencing (Ion Torrent) or targeted capture massively parallel sequencing (Illumina). Clonal mutation frequencies were estimated from the mutant allelic fractions adjusted according to tumor cellularity, tumor ploidy and local copy number states using ABSOLUTE [47]. Indel, insertion and deletion; SNV, single nucleotide variant. (B) Diagram illustrating the cancer cell fraction, as defined by ABSOLUTE, of mutations identified in cases T6, T11 and T12. Note the presence of subclonal mutations in the HER2-negative components of all cases, and in the HER2-positive components of cases T6 and T12. (C) Allelic fractions of mutations identified in HER2-positive and HER2-negative components of HER2 heterogeneous breast cancers obtained through targeted massively parallel sequencing analysis using a panel of 273 genes comprising genes frequently mutated in breast cancer and DNA repair-related genes. Indel, insertion and deletion; SNV, single nucleotide variant.
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Fig5: Sequencing analysis of HER2-positive and HER2-negative components of HER2 heterogeneous breast cancers identified founder genetic events and intra-tumor mutational heterogeneity. (A) Clonal frequencies of mutations identified in HER2-positive and HER2-negative components of HER2 heterogeneous breast cancers T6, T11 and T12, which were subjected to whole exome sequencing and orthogonal validation by amplicon sequencing (Ion Torrent) or targeted capture massively parallel sequencing (Illumina). Clonal mutation frequencies were estimated from the mutant allelic fractions adjusted according to tumor cellularity, tumor ploidy and local copy number states using ABSOLUTE [47]. Indel, insertion and deletion; SNV, single nucleotide variant. (B) Diagram illustrating the cancer cell fraction, as defined by ABSOLUTE, of mutations identified in cases T6, T11 and T12. Note the presence of subclonal mutations in the HER2-negative components of all cases, and in the HER2-positive components of cases T6 and T12. (C) Allelic fractions of mutations identified in HER2-positive and HER2-negative components of HER2 heterogeneous breast cancers obtained through targeted massively parallel sequencing analysis using a panel of 273 genes comprising genes frequently mutated in breast cancer and DNA repair-related genes. Indel, insertion and deletion; SNV, single nucleotide variant.
Mentions: To determine if the constellations of mutations would be distinct between the HER2-positive and HER2-negative components of HER2 heterogeneous breast cancers, and to identify potential driver mutations restricted to the HER2-negative components, we subjected the HER2-positive and HER2-negative components of three cases (that is, T6, T11 and T12), for which sufficient DNA from frozen tumor and matched normal tissues was available, to whole exome sequencing. Selected somatic mutations identified were validated by high-depth amplicon sequencing (Ion Torrent, 4000×) or targeted massively parallel sequencing (Figure 5A; Additional files 8, 9 and 10). Analysis of the clonal frequencies using ABSOLUTE [47] revealed that known founder genetic events such as somatic mutations in TP53 and/or PIK3CA were shared and inferred to be present in all cells of both the HER2-positive and HER2-negative components of these cases (Table 1; Figure 5A). This analysis also revealed the presence of subclonal mutations in the HER2-negative components of all cases, and in the HER2-positive component of cases T6 and T12 (Figure 5A,B). We next performed targeted massively parallel sequencing, using a panel of 273 genes frequently mutated in breast cancer and DNA repair-related genes [48], of the HER2-negative and HER2-positive components of 5 HER2 heterogeneous breast cancers (that is, T1, T3, T4, T8, and T9; Figure 5C). Consistent with the observations made by whole exome sequencing analysis, in all cases, the HER2-negative and HER2-positive components harbored somatic mutations in common, including TP53 somatic mutations in three cases (Figure 5C). Interestingly, in the two TP53 wild-type cases subjected to targeted sequencing, we identified an ARID1A mutation (that is, T4) and PIK3CA and CBFB mutations (that is, T8), which were common to the two components (Figure 5C). Taken together, in all cases analyzed, the HER2-negative and HER2-positive components shared identical somatic mutations, indicating their clonal relatedness.Figure 5

Bottom Line: HER2 is overexpressed and amplified in approximately 15% of invasive breast cancers, and is the molecular target and predictive marker of response to anti-HER2 agents.In a subset of these cases, heterogeneous distribution of HER2 gene amplification can be found, which creates clinically challenging scenarios.Our results indicate that even driver genetic alterations, such as HER2 gene amplification, can be heterogeneously distributed within a cancer, and that the HER2-negative components are likely driven by genetic alterations not present in the HER2-positive components, including BRF2 and DSN1 amplification and HER2 somatic mutations.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA. ngk1@mskcc.org.

ABSTRACT

Background: HER2 is overexpressed and amplified in approximately 15% of invasive breast cancers, and is the molecular target and predictive marker of response to anti-HER2 agents. In a subset of these cases, heterogeneous distribution of HER2 gene amplification can be found, which creates clinically challenging scenarios. Currently, breast cancers with HER2 amplification/overexpression in just over 10% of cancer cells are considered HER2-positive for clinical purposes; however, it is unclear as to whether the HER2-negative components of such tumors would be driven by distinct genetic alterations. Here we sought to characterize the pathologic and genetic features of the HER2-positive and HER2-negative components of breast cancers with heterogeneous HER2 gene amplification and to define the repertoire of potential driver genetic alterations in the HER2-negative components of these cases.

Results: We separately analyzed the HER2-negative and HER2-positive components of 12 HER2 heterogeneous breast cancers using gene copy number profiling and massively parallel sequencing, and identified potential driver genetic alterations restricted to the HER2-negative cells in each case. In vitro experiments provided functional evidence to suggest that BRF2 and DSN1 overexpression/amplification, and the HER2 I767M mutation may be alterations that compensate for the lack of HER2 amplification in the HER2-negative components of HER2 heterogeneous breast cancers.

Conclusions: Our results indicate that even driver genetic alterations, such as HER2 gene amplification, can be heterogeneously distributed within a cancer, and that the HER2-negative components are likely driven by genetic alterations not present in the HER2-positive components, including BRF2 and DSN1 amplification and HER2 somatic mutations.

No MeSH data available.


Related in: MedlinePlus