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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

HER2 heterogeneous breast cancers are ER-positive and preferentially TP53 mutant. (A) Clinico-pathologic characteristics of HER2 heterogeneous breast cancers included in this study. ER, estrogen receptor; PR progesterone receptor; WT, wild-type. (B) Micrographs of representative hematoxylin and eosin (H&E) stained sections, HER2 immunohistochemistry (IHC) and HER2 chromogenic in situ hybridization (CISH) of selected HER2 heterogeneous breast cancers included in this study (scale bar IHC, 200 μm; scale bar CISH, 50 μm). Chromosome 17 plots of microdissected HER2-positive and HER2-negative components of each case confirming the presence and absence of HER2 gene amplification (17q12), respectively. In the chromosome plots, the circular binary segmentation (cbs)-smoothed Log2 ratios for each bacterial artificial chromosome mapping to chromosome 17 were plotted on the y-axis and their genomic positions were plotted on the x-axis. Gains, amplifications and losses are highlighted in dark green, bright green and red, respectively. Please see Additional file 2 for the remaining cases.
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Fig1: HER2 heterogeneous breast cancers are ER-positive and preferentially TP53 mutant. (A) Clinico-pathologic characteristics of HER2 heterogeneous breast cancers included in this study. ER, estrogen receptor; PR progesterone receptor; WT, wild-type. (B) Micrographs of representative hematoxylin and eosin (H&E) stained sections, HER2 immunohistochemistry (IHC) and HER2 chromogenic in situ hybridization (CISH) of selected HER2 heterogeneous breast cancers included in this study (scale bar IHC, 200 μm; scale bar CISH, 50 μm). Chromosome 17 plots of microdissected HER2-positive and HER2-negative components of each case confirming the presence and absence of HER2 gene amplification (17q12), respectively. In the chromosome plots, the circular binary segmentation (cbs)-smoothed Log2 ratios for each bacterial artificial chromosome mapping to chromosome 17 were plotted on the y-axis and their genomic positions were plotted on the x-axis. Gains, amplifications and losses are highlighted in dark green, bright green and red, respectively. Please see Additional file 2 for the remaining cases.

Mentions: We identified 41 HER2-positive breast carcinomas with heterogeneous HER2 overexpression and HER2 gene amplification, of which 13 cases were amenable to microdissection. For 12 HER2 heterogeneous breast cancers the HER2-positive and HER2-negative components were separated without cross-contamination as confirmed by array-based comparative genomic hybridization (aCGH; Figure 1; Additional files 1 and 2). Histologic and immunohistochemical analysis revealed that all HER2 heterogeneous breast cancers subjected to microdissection were ER-positive (as defined by the current cutoff of >1% of ER-positive cells [25] in the whole tumor), and eight cases were also progesterone receptor (PR)-positive (Figure 1A; Additional file 1). The frequency of ER expression in these HER2 heterogeneous cases was significantly higher than that found in the HER2-positive breast cancers included in The Cancer Genome Atlas (TCGA; 56/79, 71%) dataset [10], the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) discovery (40/72, 56%) and validation (24/61, 39%) datasets [26], and the 1 year adjuvant trastuzumab versus observation for HER2-positive breast cancer (HERA) trial [27] cohort (1,536/3,383, 45%) (Fisher’s exact test, two-tailed, P = 0.0326, P = 0.0026, P < 0.0001, P < 0.0001, respectively). To define whether this observation related to the ER status of HER2 heterogeneous breast cancers would be generalizable, we investigated the ER status of the HER2 heterogeneous breast cancers that were retrieved for this study but not amenable to microdissection. The diagnostic ER immunohistochemical slides of 26 of the remaining 29 cases were retrieved, and re-analysis of their ER status revealed that 24 of 26 cases (92%) were ER-positive. The majority of the HER2 heterogeneous breast cancers in this study were of high histologic grade (9/12 grade 3, 75%, Additional file 1). Sanger sequencing revealed that all but three cases (75%) were TP53 mutant (Figure 1A; Additional file 1), a frequency similar to that found in HER2-positive breast cancers from the TCGA dataset. When assessing the HER2-positive and HER2-negative components separately, we noted that in 10 out of 12 of the HER2 heterogeneous breast cancers the ER status and histologic grade were identical between the two components of a given case, as was the TP53 status in all cases (Figure 1A; Additional file 1). Taken together, the HER2 heterogeneous breast cancers amenable to microdissection and included in this study were ER-positive and preferentially TP53 mutant.Figure 1


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)

HER2 heterogeneous breast cancers are ER-positive and preferentially TP53 mutant. (A) Clinico-pathologic characteristics of HER2 heterogeneous breast cancers included in this study. ER, estrogen receptor; PR progesterone receptor; WT, wild-type. (B) Micrographs of representative hematoxylin and eosin (H&E) stained sections, HER2 immunohistochemistry (IHC) and HER2 chromogenic in situ hybridization (CISH) of selected HER2 heterogeneous breast cancers included in this study (scale bar IHC, 200 μm; scale bar CISH, 50 μm). Chromosome 17 plots of microdissected HER2-positive and HER2-negative components of each case confirming the presence and absence of HER2 gene amplification (17q12), respectively. In the chromosome plots, the circular binary segmentation (cbs)-smoothed Log2 ratios for each bacterial artificial chromosome mapping to chromosome 17 were plotted on the y-axis and their genomic positions were plotted on the x-axis. Gains, amplifications and losses are highlighted in dark green, bright green and red, respectively. Please see Additional file 2 for the remaining cases.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4440518&req=5

Fig1: HER2 heterogeneous breast cancers are ER-positive and preferentially TP53 mutant. (A) Clinico-pathologic characteristics of HER2 heterogeneous breast cancers included in this study. ER, estrogen receptor; PR progesterone receptor; WT, wild-type. (B) Micrographs of representative hematoxylin and eosin (H&E) stained sections, HER2 immunohistochemistry (IHC) and HER2 chromogenic in situ hybridization (CISH) of selected HER2 heterogeneous breast cancers included in this study (scale bar IHC, 200 μm; scale bar CISH, 50 μm). Chromosome 17 plots of microdissected HER2-positive and HER2-negative components of each case confirming the presence and absence of HER2 gene amplification (17q12), respectively. In the chromosome plots, the circular binary segmentation (cbs)-smoothed Log2 ratios for each bacterial artificial chromosome mapping to chromosome 17 were plotted on the y-axis and their genomic positions were plotted on the x-axis. Gains, amplifications and losses are highlighted in dark green, bright green and red, respectively. Please see Additional file 2 for the remaining cases.
Mentions: We identified 41 HER2-positive breast carcinomas with heterogeneous HER2 overexpression and HER2 gene amplification, of which 13 cases were amenable to microdissection. For 12 HER2 heterogeneous breast cancers the HER2-positive and HER2-negative components were separated without cross-contamination as confirmed by array-based comparative genomic hybridization (aCGH; Figure 1; Additional files 1 and 2). Histologic and immunohistochemical analysis revealed that all HER2 heterogeneous breast cancers subjected to microdissection were ER-positive (as defined by the current cutoff of >1% of ER-positive cells [25] in the whole tumor), and eight cases were also progesterone receptor (PR)-positive (Figure 1A; Additional file 1). The frequency of ER expression in these HER2 heterogeneous cases was significantly higher than that found in the HER2-positive breast cancers included in The Cancer Genome Atlas (TCGA; 56/79, 71%) dataset [10], the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) discovery (40/72, 56%) and validation (24/61, 39%) datasets [26], and the 1 year adjuvant trastuzumab versus observation for HER2-positive breast cancer (HERA) trial [27] cohort (1,536/3,383, 45%) (Fisher’s exact test, two-tailed, P = 0.0326, P = 0.0026, P < 0.0001, P < 0.0001, respectively). To define whether this observation related to the ER status of HER2 heterogeneous breast cancers would be generalizable, we investigated the ER status of the HER2 heterogeneous breast cancers that were retrieved for this study but not amenable to microdissection. The diagnostic ER immunohistochemical slides of 26 of the remaining 29 cases were retrieved, and re-analysis of their ER status revealed that 24 of 26 cases (92%) were ER-positive. The majority of the HER2 heterogeneous breast cancers in this study were of high histologic grade (9/12 grade 3, 75%, Additional file 1). Sanger sequencing revealed that all but three cases (75%) were TP53 mutant (Figure 1A; Additional file 1), a frequency similar to that found in HER2-positive breast cancers from the TCGA dataset. When assessing the HER2-positive and HER2-negative components separately, we noted that in 10 out of 12 of the HER2 heterogeneous breast cancers the ER status and histologic grade were identical between the two components of a given case, as was the TP53 status in all cases (Figure 1A; Additional file 1). Taken together, the HER2 heterogeneous breast cancers amenable to microdissection and included in this study were ER-positive and preferentially TP53 mutant.Figure 1

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