Target Discovery and Validation Reviews and Protocols

98 Sørlie
are not internally heterogeneous, when millions of cells are sampled. The finding
that the two primary tumors and the corresponding metastases were similar in
their overall pattern of gene expression suggests that the molecular program of a
primary tumor may be retained in its metastases. This idea has been explored by
other investigators and suggests that metastatic capacity is an intrinsic feature of
the primary tumor and that the fate of the tumor might have been programmed at
an early stage of the disease (21). Hence, specific expression patterns in the primary
tumors may help identifying genes, pathways, or both that are important for
the metastatic process and for tumor progression in general.
3.2. Subclassification
To explore the possibilities for further refining these distinctions among subtypes
of breast tumors, we took advantage of the paired tumor samples. The
specific features of a gene expression pattern that are to be used as the basis for
classifying tumors should be similar in any sample taken from the same tumor,
and they should vary among different tumors. The paired samples therefore provided
a unique opportunity for a deliberate and systematic search for genes
whose expression levels reflected such intrinsic characteristics of the tumors.
Using well measured expression data from the paired samples, a subset of genes
termed “intrinsic gene set” was selected that consisted of genes with significantly
greater variation in expression between different tumors than between
paired samples from the same tumor (14). The rationale behind this list of genes
is that it is enriched for those genes whose expression patterns were characteristic
of each tumor as opposed to those that varied as a function of tissue sampling;
hence, they would be ideally suited for classification.
Our extended analyses have since included an intrinsic gene set of 540 genes
selected from expression data of 45 tumor pairs (including two primary lymph
node pairs) and approx 8000 common genes for all samples (22,23). Together,
122 tissue samples were included in the clustering analysis: 115 carcinomas
and 7 nonmalignant tissues. Most of the tumors were sampled as part of two
independent studies evaluating response to chemotherapy of locally advanced
breast cancer in an adjuvant setting. From the first cohort of patients treated
with doxorubicin monotherapy (19), 55 tumor samples were analyzed, and
from the second cohort of patients treated with 5-fluorouracil and mitomycin C
(20), 34 tumor samples were analyzed. The remaining 26 samples were primary
tumor specimens collected either at Stanford or in Norway.
The overall expression patterns showed that the tumors could be classified
into five distinct subtypes, and the main distinction was between tumors that
expressed genes characteristic of luminal epithelial cells (two groups), including
the estrogen receptor (ER) and those that were negative for these genes (three
groups) (Fig. 3). Among the luminal epithelial-type tumors, the largest group,