Principle of tumor progression tree reconstruction. (a) CGH log ratio profiles of two bladder tumors from the same patient, with color code as follows: homozygous deletions in blue, losses in green, normal regions in yellow, and gains in red. Chromosomes are delineated by gray vertical lines and a schematic representation of chromosomes and centromeres is drawn below each profile. Chromosome breakpoints common to both samples are indicated by dashed lines, with an arrow representing the sign of each breakpoint. For greater clarity, the common breakpoints on either side of the one-BAC homozygous deletion at 9p21 are not drawn. This common aberration is instead circled in each profile. (b) Tumor progression tree reconstructed for the two samples. Common breakpoints define early aberrations occurring in the common precursor of the two samples. Chromosome aberrations specific to each tumor are placed on subsequent edges.

Evaluation of the performance of TuMult and the parsimony method with simulated data. Simulated data were generated to evaluate the performance of TuMult and the parsimony method for the reconstruction of tumor progression trees. The performance of each algorithm was assessed under each set of conditions by generating 1,000 random trees and calculating (a) the percentage of the reconstructed trees with the correct topology, and (b), for the trees with the correct topology, the percentage of probes with incorrect copy number status in the internal nodes. Simulations were carried out for different numbers of tumors (upper panel), various levels of noise in the data (middle panel), and various proportions of normal cells in the samples (bottom panel). The number of tumors analyzed (between 2 and 6), together with the levels of noise (between 0.03 and 0.11) and contamination (10 to 40%) estimated for our experimental data are represented by yellow areas.

Accumulation of chromosome aberrations during breast cancer progression. Fifteen pairs of primary breast carcinomas and ipsilateral recurrences were analyzed with the TuMult algorithm. Patients are denoted as in the original article by Bollet et al. [18]. (a) In patient P14, all the aberrations of the primary tumor were found in the recurrence, consistent with a linear evolution. (b) In patient P13, both the primary tumor and the recurrence display specific events, implying that the recurrence was not directly descended from the primary tumor. (c) The proportion of all the aberrations in the tree occurring before the common precursor (white), between the common precursor and the primary tumor (blue hatched) or between the common precursor and the recurrence (red hatched) is presented for each of the 15 patients. P14 is the only example of linear evolution among the 15 trees. (d) Boxplots of the number of aberrations occurring at each step in tumor progression trees. CP, in the common precursor; PT, between the common precursor and the primary tumor; IR, between the common precursor and the ipsilateral recurrence. **P-value < 0.01, as determined in a two-tailed paired t-test.

Metastases spreading in a patient with prostate cancer. Five metastases were removed from patient 21 (data from Liu et al. [19]), including three liver metastases (21-2a, 21-2b and 21-2c), one adrenal metastasis (21-3) and one bone metastasis (21-6). (a) Tumor progression tree reconstructed with the TuMult algorithm. The three liver metastases have a longer clonal relationship than the other metastases, all being derived from common precursor 3. (b) Copy number profiles of chromosome 2 in the five metastases. Four common aberrations, delimited by dashed lines, occurred in the common precursor of all the samples. The seven circled aberrations are specific to the three liver metastases, showing these tumors diverged later in the tumor progression tree. These aberrations appeared in common precursor 3, from which the liver metastases are derived.

Overview of the TuMult algorithm. (a) Discretized copy number profiles of three tumors from the same patient (yellow, 'normal copy number'; green, 'loss'; red, 'gain'). The eight breakpoints identified in the samples (dashed lines) divide the chromosome into seven 'homogeneous segments', A to G, in which copy number is constant in any sample. The profiles can be represented as amplitude vectors (see Materials and methods), in which 'up' and 'down' breakpoints are distinguished by their position in the vector. A common breakpoint (gray shading) appears as a non-zero value at the same position in the amplitude matrix. The frequency F_k of each breakpoint is calculated from a reference data set of independent samples. (b) An identical breakpoint score (IBS), characterizing the similarity of two profiles in terms of chromosome breakpoints, is calculated for each pair of samples, and the pair displaying the highest level of similarity is selected. (c) The copy number profile of the common precursor of the two samples is reconstructed based on their common breakpoints, represented by dashed lines and black arrows. Edges are added between the common precursor (CP) and the two nodes, labeled with the aberrations defined by their specific breakpoints, represented by gray arrows. Note that a breakpoint may be both common and specific, if its amplitude is larger in one of the samples, like the 'down' breakpoint between segments A and B in this example. (d) Steps (b) and (c) are iterated until there is only one node left in the front. (e) A 'normal cell' node has been added above the common ancestor of all tumors.

Bladder tumor progression trees reconstructed with the TuMult algorithm. Thirteen samples from five patients were analyzed with the TuMult algorithm to reconstruct the tumor lineage and sequence of chromosomal aberrations in each case. Aberrations are annotated as follows: (--) homozygous deletions, (-) losses, (+) gains, (++) amplicons. Aberration boundaries are indicated in terms of chromosome cytobands. Tumor progression trees with aberrations indicated in terms of homogeneous segments are available, together with the segment description tables, from the TuMult web page [43]. Losses of chromosome arms 9q and 11p are underlined, along with homozygous deletions of 9p21.3. The aberrations -9q and -11p were the most frequent early events in the tumor progression trees. In addition, -9q and -11p occurred together on the same edge significantly more frequently than would be expected by chance (P = 0.0025). This was also true of -11p and -9p21.3 (P = 0.012). Clinical details for each sample can be found in Table 1.

Frequency of gains and losses in the metastatic precursor clones of 14 patients with metastatic prostate cancer. Fourteen patients with various metastatic samples taken from different anatomic sites were analyzed with the TuMult algorithm to generate the lineage of the metastases and to reconstruct the copy number profile of the common precursor of all metastases in each patient. The frequency of gains (in red) and losses (in green) in the genome were calculated for these 14 metastatic precursor clones.