BIOINFORMATICS APPLICATIONS NOTE
Vol. 27 no. 12 2011, pages 1698–1699
Clonality: an R package for testing clonal relatedness of two
tumors from the same patient based on their genomic profiles
Irina Ostrovnaya1,∗, Venkatraman E. Seshan1, Adam B. Olshen2and Colin B. Begg1
1Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY 10065 and
2Department of Epidemiology and Biostatistics and Helen Diller Family Comprehensive Cancer Center, University of
California—San Francisco, San Francisco, CA 94158, USA
Associate Editor: Dmitrij Frishman
Advance Access publication May 5, 2011
Summary: If a cancer patient develops multiple tumors, it is
sometimes impossible to determine whether these tumors are
independent or clonal based solely on pathological characteristics.
Investigators have studied how to improve this diagnostic challenge
by comparing the presence of loss of heterozygosity (LOH) at
selected genetic locations of tumor samples, or by comparing
genomewide copy number array profiles. We have previously
developed statistical methodology to compare such genomic profiles
for an evidence of clonality. We assembled the software for these
tests in a new R package called ‘Clonality’. For LOH profiles, the
package contains significance tests. The analysis of copy number
profiles includes a likelihood ratio statistic and reference distribution,
as well as an option to produce various plots that summarize the
release/bioc/html/Clonality.html) and http://www.mskcc.org/mskcc/
Received on December 3, 2010; revised on April 4, 2011; accepted
on April 19, 2011
Multiple cancerous lesions in the same patient can be either clonal
or independent. Clonal tumors, for example, a primary tumor and
its metastasis, originate from the same ‘clonal’ cell. A second
primary tumor develops completely independently from the original
primary. This distinction can have important clinical implications.
For example, a lung nodule in a survivor with a previous head/neck
cancer is potentially curable by surgery, if it is a primary lung
cancer, but not if it is a metastasis from the original primary. In such
cases, the genomic profiles of tumors can provide insight into their
relationship, since clonal tumors necessarily possess at least some
identical somatic mutations. Researchers have utilized two types of
genetic data for evaluating clonality. The most common approach is
loss of heterozygosity (LOH).This strategy has promise as a clinical
tool, because of its simplicity, and because it can be accomplished
easily with minimal quantities of DNA. More recently, investigators
arrays) to search for identical copy number gains or losses. This is
a more elaborate and expensive strategy, less likely to see clinical
∗To whom correspondence should be addressed.
application in the near future, but potentially valuable for its greatly
increased resolution compared with the use of candidate markers.
We have previously published statistical methodology for formal
statistical testing in both settings. For LOH data, we have developed
ratio (LR) test (Ostrovnaya et al., 2008). For CGH data, we
proposed a LR statistic and an algorithm for calculating its reference
distribution under the ‘independence’hypothesis (Ostrovnaya et al.,
2010a). We have created the R/Bioconductor (Gentleman et al.,
2004; R Development Core Team, 2010) package called ‘Clonality’
that implements these methods. This enables users to summarize
We describe the seven principal functions that are included in the
package and discuss how to interpret the output.All tests are applied
to pairs of tumors from the same patient. If multiple tumors per
patient are provided, all pairwise comparisons are performed.
The function ‘LOHclonality()’ combines both tests we have
developed for LOH data at the candidate loci (usually 10–30
markers). The main input is a matrix of LOH calls, where each
marker has to be represented by one of three user-specified symbols
denoting no LOH, LOH in allele 1 or LOH in allele 2. Markers that
are not informative (e.g. homozygous) in a particular tumor should
be given an NAinstead of a call. The methodology assumes that the
markers are independent (ideally from different chromosome arms)
and that uninformative markers are missing at random. The user
can choose to perform the CM test, the LR test or both. The test
statistic for the CM test is the number of concordant losses (i.e.
LOH on the same allele). This is printed in the output along with
counts of markers with an LOH in one tumor only, with no LOH in
both tumors and so on. The P-value is produced using a theoretical
reference distribution that relies on assumptions that alleles 1 and
2 are equally likely to be lost, and that each marker has the same
probability of an LOH. This test is valid when the deviations from
these assumptions are not substantial (Begg et al., 2007). The LR
test does not make these assumptions, but it requires knowledge
of the LOH frequencies for each marker. If they are not provided,
they can be estimated from the original dataset or another group of
reference patients with the same disease. The reference distribution
for the LR test is obtained by simulating tumors with given LOH
frequencies. Unlike the CM test, concordant LOH at an infrequently
Testing clonality using LOH data
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occurring marker can provide stronger evidence for clonality than
at a commonly occurring marker. The LR test, while free of the
assumptions of the CM test, is preferred only when the true LOH
frequencies in the specific patient population of interest are known
or can be estimated with high confidence.
For the analysis of CNAs, the software requires the package
DNAcopy (Olshen et al., 2004;Venkatraman et al., 2007).The input
into the main function, ‘clonality.analysis()’, has to be a CNAobject
used in DNAcopy; it combines chromosome, genomic locations
and each sample’s log-ratios in columns. The chromosomes should
be split into arms, since it increases the number of independent
genomic units for the analysis. This can be done using function
‘splitChromosomes()’. The clonality analysis consists of several
Testing clonality using CGH data
• The data are segmented with the one step Circular Binary
Segmentation (CBS) algorithm that selects at most one
prominent copy number change per chromosome arm (another
segmentation algorithm can potentially be used with a user-
defined segmentation function instead of the built-in ‘oneseg’,
as long as it detects at most one copy number change per
chromosome arm. Details are given in the vignette).
• The chromosome arms are classified as gain/loss/normal based
on the central or most outstanding segment. Number of Median
Absolute Deviations (MADs) of the residuals, selected by the
user, defines the gain/loss threshold. Users should investigate
the plots of the segmentation to make sure that the large
visible gains and losses pass the MAD criteria in most samples
and adjust number of MADs (‘nmad’) parameter accordingly.
Frequencies of gain or loss for each chromosome arm are
needed for the calculation of the LRs. The frequencies are
evaluated based on the original dataset, but there is also an
option to estimate them based on a dataset of other patients
with the same disease.
of the gain/loss/normal profiles. The final statistic, LR2, is the
product of LR1 and LRs from each chromosome arm in which
concordant gains or losses are observed in the two tumors.
These reflect the odds that the chromosome arm specific
mutation is clonal. LR2 quantifies the odds that the two tumors
• The reference distribution (option in ‘clonality.analysis’) is
which are necessarily independent. This distribution is used
to calculate P-values for the original tumor pairs. It is a
preferred option to the hierarchial clustering method often used
in practice, since it effectively uses the known paired structure
of the data and for other technical reasons (Ostrovnaya et al.,
2010b). Note that calculating the reference distribution might
take a substantial amount of time (hours) depending on the
number of patients and the resolution of the assay. However,
we recommend choosing this option unless the clonality signal
is very strong and the noise level is small.
It is important to make sure that small gains and losses that
could potentially be germline copy number variants (CNVs) are
excluded from the arrays prior to clonality analysis. These appear
as pronounced changes that are exactly the same in both tumors,
and are thus mistaken to be strong evidence toward clonality by our
algorithm. In order to exclude CNVs, the arrays should be either
compared to their matching normal arrays, or to the database of
genomic variants (http://projects.tcag.ca/variation/), or some other
screening method can be applied (e.g. Ostrovnaya et al., 2010c).
Since only one genomic change per chromosome arm is allowed,
we suggest limiting the resolution of the array to roughly a total of
5000–15000 markers. If the array has more markers, then mutually
exclusive blocks of several adjacent markers can be averaged using
the function ‘ave.adj.probes()’. The number of markers averaged
should be calculated based on the original resolution of the array.
Averaging also helps reduce noise in the arrays, it removes waves in
log-ratios that can result in false genomic changes, and it simplifies
the copy number patterns. If the user is not confident that all
CNVs are removed, then a lower resolution (e.g. 2000 markers)
The help file for ‘clonality.analysis()’ contains a real data
example—analysis of pairs of breast cancer lesions published by
(Hwang et al., 2004) and available from the authors’ web site.
Two functions, ‘chromosomePlots()’ and ‘genomewidePlots()’,
provide per chromosome and genomewide plots of the data and
one step segmentation for pairs of tumors. These plots show where
the clonality signal is coming from; they illustrate the concordance
between general mutational patterns and the match between specific
gains and losses. The overlaying histograms of the LRs and the
reference distribution under the hypothesis of independence are
produced by ‘histogramPlot()’. The extent of overlap between the
two histograms can help define the cut-offs for diagnosing patients:
the upper right tail of the reference distribution might be interpreted
as an equivocal area, while the LRs above or below the equivocal
area receive unambiguous diagnoses.
Visualization of CGH data
in the development of this project.
Funding: National Cancer Institute award (CA124504).
Conflict of Interest: none declared.
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