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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

BRF2 and DSN1 amplifications are potential driver genetic alterations in HER2-negative breast cancer cells. (A) Nuclear subcellular localization of BRF2 and DSN1 in NIH3T3 (top) and MCF10A (bottom) cells expressing BRF2-ZsGreen and DSN1-ZsGreen (scale bar, 25 μm). (B) Foci formation assay of NIH3T3 cells expressing vector control, BRF2 or DSN1 protein. Cells were fixed and stained with crystal violet 21 days after plating, and the foci were quantified (see Materials and methods). *P < 0.05, unpaired t-test. Error bars represent standard deviation of mean. (C) Anchorage-independent growth of MCF10A cells expressing vector control, BRF2 or DSN1 protein. Quantification was performed using an MTT assay (left) or by defining the number and size of colonies (right). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired t-test. Error bars represent standard deviation of mean. (D) Impact of empty vector, BRF2 and DSN1 expression on growth and glandular architecture of MCF10A (top) and MCF12A (bottom) cells grown in three-dimensional basement membrane cultures (scale bar, 500 μm).
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Fig4: BRF2 and DSN1 amplifications are potential driver genetic alterations in HER2-negative breast cancer cells. (A) Nuclear subcellular localization of BRF2 and DSN1 in NIH3T3 (top) and MCF10A (bottom) cells expressing BRF2-ZsGreen and DSN1-ZsGreen (scale bar, 25 μm). (B) Foci formation assay of NIH3T3 cells expressing vector control, BRF2 or DSN1 protein. Cells were fixed and stained with crystal violet 21 days after plating, and the foci were quantified (see Materials and methods). *P < 0.05, unpaired t-test. Error bars represent standard deviation of mean. (C) Anchorage-independent growth of MCF10A cells expressing vector control, BRF2 or DSN1 protein. Quantification was performed using an MTT assay (left) or by defining the number and size of colonies (right). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired t-test. Error bars represent standard deviation of mean. (D) Impact of empty vector, BRF2 and DSN1 expression on growth and glandular architecture of MCF10A (top) and MCF12A (bottom) cells grown in three-dimensional basement membrane cultures (scale bar, 500 μm).

Mentions: From this list of genes, we focused on BRF2 and DSN1, given the lack of direct functional evidence to support a potential oncogenic role of the amplification of these genes in breast cancer. Hence, we sought to define whether BRF2 and DSN1 would have oncogenic properties in in vitro models of breast cancer. BRF2 (8p11.23) maps to the 8p11-p12 amplicon and is reported to be recurrently amplified in 10 to 15% of breast cancers [28]. This gene encodes a subunit of the RNA polymerase III transcription initiation factor and has been identified as a potential oncogene in lung squamous cell carcinomas [42]. DSN1 maps to 20q11.23, and encodes a kinetochore protein of the minichromosome instability-12 centromere complex [43]. This gene is amplified only in 1.7% of all breast cancers, and its amplification is mutually exclusive with HER2 amplification in the TCGA luminal breast cancer dataset (Figure 3B) [10]. The DSN1 amplicon is distinct from the 20q13 amplicon, and is not encompassed by its smallest region of amplification [30]. Forced expression of BRF2 and DSN1 in NIH3T3 and non-malignant MCF10A breast epithelial cells resulted in their nuclear localization as expected (Figure 4A; Additional file 7), and in significant transformation of NIH3T3 and MCF10A cells as measured by a foci formation assay (Figure 4B) and anchorage-independent growth in soft agar (Figure 4C), respectively. In addition, forced expression of BRF2 and DSN1 in non-malignant breast epithelial cells MCF10A and MCF12A affected the growth and glandular architecture of these cells when grown in three-dimensional culture systems. Whilst empty vector-transfected MCF10A and MCF12A cells formed spheroid acinar-like structures, BRF2 and DSN1 overexpression led to larger, multiacinar structures with filled lumens (Figure 4D), in line with phenotypes previously observed when oncoproteins are expressed in this model system [44,45].Figure 4


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)

BRF2 and DSN1 amplifications are potential driver genetic alterations in HER2-negative breast cancer cells. (A) Nuclear subcellular localization of BRF2 and DSN1 in NIH3T3 (top) and MCF10A (bottom) cells expressing BRF2-ZsGreen and DSN1-ZsGreen (scale bar, 25 μm). (B) Foci formation assay of NIH3T3 cells expressing vector control, BRF2 or DSN1 protein. Cells were fixed and stained with crystal violet 21 days after plating, and the foci were quantified (see Materials and methods). *P < 0.05, unpaired t-test. Error bars represent standard deviation of mean. (C) Anchorage-independent growth of MCF10A cells expressing vector control, BRF2 or DSN1 protein. Quantification was performed using an MTT assay (left) or by defining the number and size of colonies (right). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired t-test. Error bars represent standard deviation of mean. (D) Impact of empty vector, BRF2 and DSN1 expression on growth and glandular architecture of MCF10A (top) and MCF12A (bottom) cells grown in three-dimensional basement membrane cultures (scale bar, 500 μm).
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Fig4: BRF2 and DSN1 amplifications are potential driver genetic alterations in HER2-negative breast cancer cells. (A) Nuclear subcellular localization of BRF2 and DSN1 in NIH3T3 (top) and MCF10A (bottom) cells expressing BRF2-ZsGreen and DSN1-ZsGreen (scale bar, 25 μm). (B) Foci formation assay of NIH3T3 cells expressing vector control, BRF2 or DSN1 protein. Cells were fixed and stained with crystal violet 21 days after plating, and the foci were quantified (see Materials and methods). *P < 0.05, unpaired t-test. Error bars represent standard deviation of mean. (C) Anchorage-independent growth of MCF10A cells expressing vector control, BRF2 or DSN1 protein. Quantification was performed using an MTT assay (left) or by defining the number and size of colonies (right). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired t-test. Error bars represent standard deviation of mean. (D) Impact of empty vector, BRF2 and DSN1 expression on growth and glandular architecture of MCF10A (top) and MCF12A (bottom) cells grown in three-dimensional basement membrane cultures (scale bar, 500 μm).
Mentions: From this list of genes, we focused on BRF2 and DSN1, given the lack of direct functional evidence to support a potential oncogenic role of the amplification of these genes in breast cancer. Hence, we sought to define whether BRF2 and DSN1 would have oncogenic properties in in vitro models of breast cancer. BRF2 (8p11.23) maps to the 8p11-p12 amplicon and is reported to be recurrently amplified in 10 to 15% of breast cancers [28]. This gene encodes a subunit of the RNA polymerase III transcription initiation factor and has been identified as a potential oncogene in lung squamous cell carcinomas [42]. DSN1 maps to 20q11.23, and encodes a kinetochore protein of the minichromosome instability-12 centromere complex [43]. This gene is amplified only in 1.7% of all breast cancers, and its amplification is mutually exclusive with HER2 amplification in the TCGA luminal breast cancer dataset (Figure 3B) [10]. The DSN1 amplicon is distinct from the 20q13 amplicon, and is not encompassed by its smallest region of amplification [30]. Forced expression of BRF2 and DSN1 in NIH3T3 and non-malignant MCF10A breast epithelial cells resulted in their nuclear localization as expected (Figure 4A; Additional file 7), and in significant transformation of NIH3T3 and MCF10A cells as measured by a foci formation assay (Figure 4B) and anchorage-independent growth in soft agar (Figure 4C), respectively. In addition, forced expression of BRF2 and DSN1 in non-malignant breast epithelial cells MCF10A and MCF12A affected the growth and glandular architecture of these cells when grown in three-dimensional culture systems. Whilst empty vector-transfected MCF10A and MCF12A cells formed spheroid acinar-like structures, BRF2 and DSN1 overexpression led to larger, multiacinar structures with filled lumens (Figure 4D), in line with phenotypes previously observed when oncoproteins are expressed in this model system [44,45].Figure 4

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