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Genomic (in)stability of the breast tumor microenvironment

ABSTRACT: The breast tumor microenvironment plays an active role in tumorigenesis. Molecular alterations, including epigenetic modifications to DNA, and changes in RNA and protein expression have been identified in tumor-associated stroma; however, there is considerable debate as to whether the stroma is characterized by genomic instability or whether detection of chromosomal alterations in the breast stroma is a reflection of technological artifact rather than the true genomic content of the tumor microenvironment. Methods: Surgically-removed breast stroma specimens from 112 women undergoing reductive mammoplasty (n=15), prophylactic mastectomy (N=6) or mastectomy for a diagnosis of breast disease (n=92) were frozen in optimal cutting temperature medium. Allelic imbalance (AI) analysis was performed in 484 stromal specimens from 98 women using a panel of 52 microsatellite markers; SNP data was generated from a subset of 86 stromal specimens using 250K SNP arrays (Affymetrix). Copy number alterations were identified using Partek Genomics Suite. Results: AI was not detected in 92% (444/484) of stroma specimens. When compared to previously generated AI data from 77 formalin-fixed, paraffin-embedded stroma specimens (Ellsworth et al., Ann Surg Oncol 2004), 32 (42%) of which harbored at least one detectable AI event, the frequency of AI in the FFPE specimens (4.62%) was significantly higher (P<0.0001) than that found in frozen specimens (0.45%). Of the stroma specimens assayed using SNP arrays 95% (82/86) had no detectable alterations and the 11 copy number changes were small and not shared between specimens. Conclusions: The data presented here support a model in which the tumor microenvironment is genetically stable. The direct comparison of copy number alterations between FFPE and frozen research-grade specimens using identical methodologies suggests that past reports of significant AI in breast stroma, both adjacent to and distant from the tumor, reflects artifact in the archival specimens caused by formalin-fixation, paraffin-embedding and tissue storage.

ORGANISM(S): Homo sapiens

PROVIDER: GSE38071 | GEO | 2012/12/21

SECONDARY ACCESSION(S): PRJNA167184

REPOSITORIES: GEO

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Project description:The breast tumor microenvironment plays an active role in tumorigenesis. Molecular alterations, including epigenetic modifications to DNA, and changes in RNA and protein expression have been identified in tumor-associated stroma; however, there is considerable debate as to whether the stroma is characterized by genomic instability or whether detection of chromosomal alterations in the breast stroma is a reflection of technological artifact rather than the true genomic content of the tumor microenvironment. Methods: Surgically-removed breast stroma specimens from 112 women undergoing reductive mammoplasty (n=15), prophylactic mastectomy (N=6) or mastectomy for a diagnosis of breast disease (n=92) were frozen in optimal cutting temperature medium. Allelic imbalance (AI) analysis was performed in 484 stromal specimens from 98 women using a panel of 52 microsatellite markers; SNP data was generated from a subset of 86 stromal specimens using 250K SNP arrays (Affymetrix). Copy number alterations were identified using Partek Genomics Suite. Results: AI was not detected in 92% (444/484) of stroma specimens. When compared to previously generated AI data from 77 formalin-fixed, paraffin-embedded stroma specimens (Ellsworth et al., Ann Surg Oncol 2004), 32 (42%) of which harbored at least one detectable AI event, the frequency of AI in the FFPE specimens (4.62%) was significantly higher (P<0.0001) than that found in frozen specimens (0.45%). Of the stroma specimens assayed using SNP arrays 95% (82/86) had no detectable alterations and the 11 copy number changes were small and not shared between specimens. Conclusions: The data presented here support a model in which the tumor microenvironment is genetically stable. The direct comparison of copy number alterations between FFPE and frozen research-grade specimens using identical methodologies suggests that past reports of significant AI in breast stroma, both adjacent to and distant from the tumor, reflects artifact in the archival specimens caused by formalin-fixation, paraffin-embedding and tissue storage. 250K STY data was generated for 86 breast stromal specimens. The tissue specimens were analyzed by paired analysis to the SNP data from matched genomic (blood) DNAs

Project description:We searched the head and neck pathology database at The University of Texas MD Anderson Cancer Center (Houston, Texas) to identify all patients with SGCs who had been treated surgically from 1995 to 2008. Twenty cases from each tumor type, ACCs, MECs, and SDCs were selected based on the availability of at least 1.0 gm of fresh frozen tumor tissues. Seventeen salivary gland tissues (10 from matching tumor cases and 7 from neck resections from patients with other diseases) were used as control (Supplemental Table 1). The 20 ACCs had previously been included in our earlier comparative genomic hybridization analysis. 32 The tissues had been harvested immediately after resection by experienced head and neck pathologists, placed in liquid nitrogen, and stored at -80°C until use. Frozen sections of each tissue specimen were evaluated for tumor content and quality. Specimens with more than 20% of host non-neoplastic elements in any given sample underwent macrodissection to enrich the tumor percentage to more than 80%. One ACC tumor was disqualified because of excessive stroma; of the 59 tumor specimens, 14 had undergone mass dissection to remove non-tumor elements. All tissues were obtained according to an institutional review board-approved protocol for non-mucosal head and neck cancer. DNA extraction: Fresh-frozen tissue was processed using the Gentra Puregene tissue kit (QIAGEN, Valencia, CA). In brief, normal and tumor tissues were carefully dissected, and proteinase K was immediately added for cell digestion. After protein precipitation, DNA was precipitated using isopropanol, followed by a 70% ethanol washing procedure. Purified DNA was dissolved and eluted in DNA hydration solution and stored at -80°C. The quality of DNA was assessed using electrophoresis and nano-drop measurement. A 260/280 wavelength ratio of at least 1.8 was used to qualify specimens for analysis. Microarray analysis: We used the Affymetrix 250k Nsp SNP array (Affymetrix, Inc., Santa Clara, CA) to survey the genotype and DNA CNA copy number data for 59 tumor specimens and 17 healthy salivary gland specimens. Each array contains 261,563 SNP sites, which are approximately uniformly distributed throughout the genome. The SNP site mapping information was provided by Affymetrix, Inc., using human genome sequence version NCBI36/hg18. Two samples were repeatedly sampled to assess data reproducibility.

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Genomic (in)stability of the breast tumor microenvironment

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