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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

Identification of a HER2 mutation as a potential driver genetic alteration in the HER2-negative component of a HER2 heterogeneous breast cancer. (A) Cell-free in vitro kinase assay determining the tyrosine kinase activity of the Poly(Glu4-Tyr) substrate and the autophosphorylation activity of wild-type (WT) HER2 (dark gray) and I767M mutant HER2 (light gray) in the presence and absence of neuregulin-1 (NRG1). Tyrosine kinase activity was assessed using the ADP Hunter HS Assay (DiscoveRx, left). Western blot analysis of representative elutes post-kinase assay of HER2-tagRFP, HER2(I767M)-tagRFP and tagRFP control proteins. The amounts of HER2 wild-type and I767M mutant HER2 enzymes used in the DiscoveRx kinase assay were similar as confirmed using antibodies against total HER2 (top panel) and tagRFP (bottom panel). ****P < 0.0001, Holm-Šídák-correction, multiple t-test. Error bars represent standard deviation of mean. (B) The Tyrosine Kinase Assay Kit (Millipore) confirmed the significantly higher transphosphorylation of the tyrosine kinase substrate Poly(Glu4-Tyr) by I767M mutant HER2 (light gray) compared with wild-type HER2 (dark gray). ***P < 0.001, Holm-Šídák-correction, multiple t-test. Error bars represent standard deviation of mean. (C) Foci formation assay of NIH3T3 cells stably expressing empty vector, wild-type HER2 or I767M mutant HER2 protein. Cells were fixed and stained with crystal violet 5 and 12 days after plating. Quantification was performed at day 5. Note that at day 12, the wild-type HER2 resulted in increased foci formation. ***P < 0.001, unpaired t-test. Error bars represent standard deviation of mean. (D) Anchorage-independent growth of MCF10A cells stably expressing empty vector, wild-type HER2 or I767M mutant HER2 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 < 0.0001, unpaired t-test. N.s., not significant. Error bars represent standard deviation of mean. (E) Effect of stable expression of wild-type HER2 (blue) and I767M mutant HER2 (red) on survival and growth of ER-positive MCF7 (PIK3CA mutant) and T47D (PIK3CA and TP53 mutant) cells in growth media with or without neuregulin-1 (NRG1, 10 ng/ml). **P < 0.01, ***P < 0.001, Holm-Šídák-correction, multiple t-test. Error bars represent standard deviation of mean.
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Fig6: Identification of a HER2 mutation as a potential driver genetic alteration in the HER2-negative component of a HER2 heterogeneous breast cancer. (A) Cell-free in vitro kinase assay determining the tyrosine kinase activity of the Poly(Glu4-Tyr) substrate and the autophosphorylation activity of wild-type (WT) HER2 (dark gray) and I767M mutant HER2 (light gray) in the presence and absence of neuregulin-1 (NRG1). Tyrosine kinase activity was assessed using the ADP Hunter HS Assay (DiscoveRx, left). Western blot analysis of representative elutes post-kinase assay of HER2-tagRFP, HER2(I767M)-tagRFP and tagRFP control proteins. The amounts of HER2 wild-type and I767M mutant HER2 enzymes used in the DiscoveRx kinase assay were similar as confirmed using antibodies against total HER2 (top panel) and tagRFP (bottom panel). ****P < 0.0001, Holm-Šídák-correction, multiple t-test. Error bars represent standard deviation of mean. (B) The Tyrosine Kinase Assay Kit (Millipore) confirmed the significantly higher transphosphorylation of the tyrosine kinase substrate Poly(Glu4-Tyr) by I767M mutant HER2 (light gray) compared with wild-type HER2 (dark gray). ***P < 0.001, Holm-Šídák-correction, multiple t-test. Error bars represent standard deviation of mean. (C) Foci formation assay of NIH3T3 cells stably expressing empty vector, wild-type HER2 or I767M mutant HER2 protein. Cells were fixed and stained with crystal violet 5 and 12 days after plating. Quantification was performed at day 5. Note that at day 12, the wild-type HER2 resulted in increased foci formation. ***P < 0.001, unpaired t-test. Error bars represent standard deviation of mean. (D) Anchorage-independent growth of MCF10A cells stably expressing empty vector, wild-type HER2 or I767M mutant HER2 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 < 0.0001, unpaired t-test. N.s., not significant. Error bars represent standard deviation of mean. (E) Effect of stable expression of wild-type HER2 (blue) and I767M mutant HER2 (red) on survival and growth of ER-positive MCF7 (PIK3CA mutant) and T47D (PIK3CA and TP53 mutant) cells in growth media with or without neuregulin-1 (NRG1, 10 ng/ml). **P < 0.01, ***P < 0.001, Holm-Šídák-correction, multiple t-test. Error bars represent standard deviation of mean.

Mentions: When focusing on the mutations restricted to the HER2-negative components, we identified a HER2 I767M somatic mutation in one of the three cases subjected to whole exome sequencing (T6; Figure 5A; Additional file 11). HER2 somatic mutations have been shown not to result in HER2 overexpression using the current immunohistochemical assays [7]. The HER2 I767M kinase domain mutation has previously been reported to increase the levels of HER2 phosphorylation modestly in MCF10A cells [7], but it has not been further evaluated for its potential as an activating driver event. Here we demonstrate using two independent cell-free kinase assays that the HER2 I767M mutation displayed significantly increased transphosphorylation of the tyrosine kinase substrate Poly(Glu4-Tyr) compared with wild-type HER2 (Figure 6A,B). We next investigated whether the HER2 I767M mutation would result in transformation of NIH3T3 cells. Stable forced expression of the HER2 I767M mutation resulted in significantly increased foci formation compared with empty vector and wild-type HER2 (Figure 6C). To define the impact of the HER2 I767M mutation on anchorage-independent growth, we generated MCF10A cells stably expressing the empty vector, wild-type HER2 and the HER2 I767M mutation. Soft agar assays revealed that both wild-type and I767M mutant HER2 led to an increase in the number of colonies compared with empty vector; however, the HER2 I767M mutation resulted in significantly larger colonies than those caused by wild-type HER2 (Figure 6D). Similar results were obtained with NIH3T3 cells stably expressing the empty vector, wild-type HER2 and the HER2 I767M mutation (Additional file 12). To define whether the HER2 I767M mutation would have an impact similar to that of HER2 tyrosine kinase mutations previously shown to be strongly activating (V777L) or not activating (Y835F) by Bose et al. [7], we transiently forced the expression of empty vector, wild-type HER2, HER2 I767M, HER2 V777L and HER2 Y835F in NIH3T3, MCF10A and MCF12A cells. These transiently transfected cells were subsequently subjected to a soft agar assay which demonstrated that the HER2 I767M mutation resulted in anchorage-independent growth in soft agar that was higher than that caused by wild-type HER2 and the non-activating HER2 mutation (Y835F), but not statistically different from that caused by the strongly activating mutation (V777L) (Additional file 13).Figure 6


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)

Identification of a HER2 mutation as a potential driver genetic alteration in the HER2-negative component of a HER2 heterogeneous breast cancer. (A) Cell-free in vitro kinase assay determining the tyrosine kinase activity of the Poly(Glu4-Tyr) substrate and the autophosphorylation activity of wild-type (WT) HER2 (dark gray) and I767M mutant HER2 (light gray) in the presence and absence of neuregulin-1 (NRG1). Tyrosine kinase activity was assessed using the ADP Hunter HS Assay (DiscoveRx, left). Western blot analysis of representative elutes post-kinase assay of HER2-tagRFP, HER2(I767M)-tagRFP and tagRFP control proteins. The amounts of HER2 wild-type and I767M mutant HER2 enzymes used in the DiscoveRx kinase assay were similar as confirmed using antibodies against total HER2 (top panel) and tagRFP (bottom panel). ****P < 0.0001, Holm-Šídák-correction, multiple t-test. Error bars represent standard deviation of mean. (B) The Tyrosine Kinase Assay Kit (Millipore) confirmed the significantly higher transphosphorylation of the tyrosine kinase substrate Poly(Glu4-Tyr) by I767M mutant HER2 (light gray) compared with wild-type HER2 (dark gray). ***P < 0.001, Holm-Šídák-correction, multiple t-test. Error bars represent standard deviation of mean. (C) Foci formation assay of NIH3T3 cells stably expressing empty vector, wild-type HER2 or I767M mutant HER2 protein. Cells were fixed and stained with crystal violet 5 and 12 days after plating. Quantification was performed at day 5. Note that at day 12, the wild-type HER2 resulted in increased foci formation. ***P < 0.001, unpaired t-test. Error bars represent standard deviation of mean. (D) Anchorage-independent growth of MCF10A cells stably expressing empty vector, wild-type HER2 or I767M mutant HER2 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 < 0.0001, unpaired t-test. N.s., not significant. Error bars represent standard deviation of mean. (E) Effect of stable expression of wild-type HER2 (blue) and I767M mutant HER2 (red) on survival and growth of ER-positive MCF7 (PIK3CA mutant) and T47D (PIK3CA and TP53 mutant) cells in growth media with or without neuregulin-1 (NRG1, 10 ng/ml). **P < 0.01, ***P < 0.001, Holm-Šídák-correction, multiple t-test. Error bars represent standard deviation of mean.
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Fig6: Identification of a HER2 mutation as a potential driver genetic alteration in the HER2-negative component of a HER2 heterogeneous breast cancer. (A) Cell-free in vitro kinase assay determining the tyrosine kinase activity of the Poly(Glu4-Tyr) substrate and the autophosphorylation activity of wild-type (WT) HER2 (dark gray) and I767M mutant HER2 (light gray) in the presence and absence of neuregulin-1 (NRG1). Tyrosine kinase activity was assessed using the ADP Hunter HS Assay (DiscoveRx, left). Western blot analysis of representative elutes post-kinase assay of HER2-tagRFP, HER2(I767M)-tagRFP and tagRFP control proteins. The amounts of HER2 wild-type and I767M mutant HER2 enzymes used in the DiscoveRx kinase assay were similar as confirmed using antibodies against total HER2 (top panel) and tagRFP (bottom panel). ****P < 0.0001, Holm-Šídák-correction, multiple t-test. Error bars represent standard deviation of mean. (B) The Tyrosine Kinase Assay Kit (Millipore) confirmed the significantly higher transphosphorylation of the tyrosine kinase substrate Poly(Glu4-Tyr) by I767M mutant HER2 (light gray) compared with wild-type HER2 (dark gray). ***P < 0.001, Holm-Šídák-correction, multiple t-test. Error bars represent standard deviation of mean. (C) Foci formation assay of NIH3T3 cells stably expressing empty vector, wild-type HER2 or I767M mutant HER2 protein. Cells were fixed and stained with crystal violet 5 and 12 days after plating. Quantification was performed at day 5. Note that at day 12, the wild-type HER2 resulted in increased foci formation. ***P < 0.001, unpaired t-test. Error bars represent standard deviation of mean. (D) Anchorage-independent growth of MCF10A cells stably expressing empty vector, wild-type HER2 or I767M mutant HER2 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 < 0.0001, unpaired t-test. N.s., not significant. Error bars represent standard deviation of mean. (E) Effect of stable expression of wild-type HER2 (blue) and I767M mutant HER2 (red) on survival and growth of ER-positive MCF7 (PIK3CA mutant) and T47D (PIK3CA and TP53 mutant) cells in growth media with or without neuregulin-1 (NRG1, 10 ng/ml). **P < 0.01, ***P < 0.001, Holm-Šídák-correction, multiple t-test. Error bars represent standard deviation of mean.
Mentions: When focusing on the mutations restricted to the HER2-negative components, we identified a HER2 I767M somatic mutation in one of the three cases subjected to whole exome sequencing (T6; Figure 5A; Additional file 11). HER2 somatic mutations have been shown not to result in HER2 overexpression using the current immunohistochemical assays [7]. The HER2 I767M kinase domain mutation has previously been reported to increase the levels of HER2 phosphorylation modestly in MCF10A cells [7], but it has not been further evaluated for its potential as an activating driver event. Here we demonstrate using two independent cell-free kinase assays that the HER2 I767M mutation displayed significantly increased transphosphorylation of the tyrosine kinase substrate Poly(Glu4-Tyr) compared with wild-type HER2 (Figure 6A,B). We next investigated whether the HER2 I767M mutation would result in transformation of NIH3T3 cells. Stable forced expression of the HER2 I767M mutation resulted in significantly increased foci formation compared with empty vector and wild-type HER2 (Figure 6C). To define the impact of the HER2 I767M mutation on anchorage-independent growth, we generated MCF10A cells stably expressing the empty vector, wild-type HER2 and the HER2 I767M mutation. Soft agar assays revealed that both wild-type and I767M mutant HER2 led to an increase in the number of colonies compared with empty vector; however, the HER2 I767M mutation resulted in significantly larger colonies than those caused by wild-type HER2 (Figure 6D). Similar results were obtained with NIH3T3 cells stably expressing the empty vector, wild-type HER2 and the HER2 I767M mutation (Additional file 12). To define whether the HER2 I767M mutation would have an impact similar to that of HER2 tyrosine kinase mutations previously shown to be strongly activating (V777L) or not activating (Y835F) by Bose et al. [7], we transiently forced the expression of empty vector, wild-type HER2, HER2 I767M, HER2 V777L and HER2 Y835F in NIH3T3, MCF10A and MCF12A cells. These transiently transfected cells were subsequently subjected to a soft agar assay which demonstrated that the HER2 I767M mutation resulted in anchorage-independent growth in soft agar that was higher than that caused by wild-type HER2 and the non-activating HER2 mutation (Y835F), but not statistically different from that caused by the strongly activating mutation (V777L) (Additional file 13).Figure 6

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