Illumina Genome Analyzer IIx sequencing; GSM1150560: STT5424_Breast_001_EN; Homo sapiens; RNA-Seq

STT5424_Breast_001_EN

The earliest recognizable stages of breast neoplasia are lesions that represent a heterogeneous collection of epithelial proliferations currently classified based on morphology. Their role in the development of breast cancer is not well understood but insight into the critical events at this early stage will improve efforts in breast cancer detection and prevention. These microscopic lesions are technically difficult to study so very little is known about their molecular alterations.  To characterize the transcriptional changes of early breast neoplasia, we sequenced 3''- end enriched RNAseq libraries from formalin-fixed paraffin-embedded tissue of early neoplasia samples and matched normal breast and carcinoma samples from 25 patients. We find that gene  expression patterns within early neoplasias are distinct from both normal and breast cancer patterns and identify a pattern of pro-oncogenic changes, including elevated transcription of ERBB2, FOXA1, and GATA3 at this early stage. We validate these findings on a second independent gene expression profile data set generated by whole transcriptome sequencing. Measurements of protein expression by immunohistochemistry on an independent set of early neoplasias confirms that ER pathway regulators FOXA1 and GATA3, as well as ER itself, are consistently upregulated at this early stage. The early neoplasia samples also demonstrate coordinated changes in long non-coding RNA expression and microenvironment stromal gene expression patterns.  This study is the first examination of global gene expression in early breast neoplasia, and the genes identified here represent candidate participants in the earliest molecular events in the development of breast cancer. Overall design: 3SEQ was performed on 72 FFPE human breast samples from 25 patients:  24 normal, 25 early neoplasia, 9 carcinoma in situ, and 14 invasive cancer

A shared transcriptional program in early breast neoplasias despite genetic and clinical distinctions

Submitted by Gene Expression Omnibus on 12-MAY-2014

Multiple 2 mm-diameter core punches were taken from FFPE tissue blocks to collect dense areas of neoplasia and adjacent normal breast epithelial content separately.  When ductal carcinoma in situ (DCIS) and/or invasive ductal carcinoma (IDC) were present, these were also cored.  Three to ten tissue cores were pooled for each diagnosis per patient, and tissue cores were re-embedded in paraffin and re-evaluated histologically for lesion purity by sectioning length-wise along the core.  Only samples which possessed >90% of luminal cells with the appropriate diagnosis within the epithelial compartment were included among our samples.  Adjacent normal cores contained no identifiable pathologies, and cancer cores possessed less than 5% early neoplasia or normal epithelial cells.  In all cases stromal cells were present in the core samples.  DNA and total RNA were purified from paraffin slices of the embedded tissue cores following deparaffination with a xylene incubation, ethanol wash, and a protease digestion, using the RecoverAll Total Nucleic Acid Isolation Kit (Ambion/Life Technologies; catalog #1975). 3SEQ libraries were prepared from as little as 5 ug total RNA.  RNA was enriched for 3’ ends by using the Oligotex mRNA mini Kit (QIAgen).  The 3’- poly(A)-enriched RNA pools were made into directional 3SEQ Illumina sequencing libraries as follows.  RNA that was not sufficiently fragmented was heat-sheared to a size of approximately 100-200 bases by incubation with First Strand Buffer (Invitrogen/Life Technologies) and oligo-dT-P7 primer (5’- CAA GCA GAA GAC GGC ATA CGA GCT CTT CCG ATC TTT TTT TTT TTT TTT TTT TTT TTT TVN -3’) at 85oC for 3-5 minutes, depending on the extent of fragmentation required.  The mixture was cooled to 50oC and first strand cDNA synthesis was performed in a 20 ul reaction using Superscript III Reverse Transcriptase (Invitrogen/Life Technologies) for 1 hour at 50oC.  Second strand cDNA was synthesized using Second Strand Buffer (Invitrogen/Life Technologies), DNA ligase (Invitrogen/Life Technologies), DNA polymerase I (New England Biolabs), and RNaseH (Epicentre Biotechnologies/Illumina), and purified using the QIAquick PCR Purification Kit (QIAgen).  3’ end repair and modification used dATP, Klenow exo (New England Biolabs) and Klenow Buffer, and was purified using the QIAgen MinElute PCR Purification Kit (QIAgen).  cDNA was then ligated to the double-stranded P5 Linker (Adapter Oligo Mix; Illumina) for 15 minutes at 22oC.  SPRI bead purification (Agencourt Biosciences/Beckman Coulter) was performed both prior to and following PCR amplification of the linker-ligated cDNA using 5' - AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T - 3' and 5' - CAA GCA GAA GAC GGC ATA CGA GCT CTT CCG ATC - 3' primers and the Phusion PCR Master Mix (New England Biolabs) using a 15 cycle program (98oC for 30 sec; 15 cycles of 98oC for 10 sec, 65oC for 30 sec, 72oC for 30 sec; 72oC for 5 min).  The libraries were then size selected for 200–300 bp fragments by agarose gel fractionation (3% Nusieve GTG; Lonza/Fisher Scientific) and purified using the QIAquick Gel Extraction Kit (QIAgen).  The libraries contain P7 sequence at the 3’ end downstream of poly(A) and P5 at the 5’ end.

A shared transcriptional program in early breast neoplasias despite genetic and clinical distinctions

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