1p36 deletion is a marker for tumour dissemination in microsatellite stable stage II-III colon cancer

The study cohort included 116 patients operated for colorectal cancer between 1985 and 2006 at the Uppsala University hospital and at Västerås district general hospital between 2000-2003, with fresh frozen tissue samples available. We aimed at selecting between 20-25 cases each with stages II and III with and without distant recurrence and stage IV. Morphological and clinical parameters were retrieved from the original pathology reports. Patients with a history of preoperative therapy or with a surgical or pathology report suggesting a non-radical resection margin (R1 or R2 resection) were excluded. To secure the quality of disease staging, patients with stage II disease were only included if at least 10 lymph nodes were analysed. Patients with disease stage II-III and no recurrence were only included if the follow-up time was longer than 5 years. Tumour cell content was required to be at least 40% in the frozen tissue block. The study design was chosen to have few factors confounding an association between the tumour genome at diagnosis (surgery) and development of distant metastasis. Clinical and histological characteristics are presented in Table 1. Adjuvant chemotherapy, chiefly with a fluoropyrimidine alone was given to 22 out of 53 stage II-III patients without recurrence and to 29 out of 40 patients in stages II-III who developed distant metastasis.

Genomic DNA was extracted from 10 μm sections of the fresh frozen tissue using QIAamp DNA mini kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s recommendations for DNA purification from tissue. Alternatively, genomic DNA was extracted from approximately 25 mg of each fresh frozen colon tissue sample on a Tecan Evo MCA 150 robotic platform using the extraction method described in Mathot et al[13]. DNA concentration was determined using NanoDrop (Thermo Scientific, Wilmington, DE).

MSI status was determined using MSI Analysis System, version 1.2 (Promega, Madison, WI) with 6 ng genomic DNA and analysis of five mononucleotide repeat markers (BAT25, BAT26, NR-21, NR-24 and MONO-27). Analyses were performed on a 3130xl genetic analyser (Applied Biosystems, Foster city, CA). According to guidelines from a National Cancer Institute workshop in 1997, samples were denoted MSI-High (MSI-H) if two or more of the five markers show instability, MSI-Low (MSI-L) if only one marker shows instability and microsatellite stable (MSS) if no markers display instability. Recent studies indicate no significant difference between MSI-L and MSS [14] and they were therefore grouped together as MSS in this study.

Array experiments were performed according to standard protocols for Affymetrix GeneChip® Mapping SNP 6.0 arrays (Affymetrix Cytogenetics Copy Number Assay User Guide (P/N 702607 Rev2.), Affymetrix Inc., Santa Clara, CA). 500 ng total genomic DNA was digested with a restriction enzyme (Nsp, Sty), ligated to an appropriate adapter for the enzyme, and subjected to PCR amplification using a single primer. After digestion with DNase I, PCR products were labelled with a biotinylated nucleotide analogue using terminal deoxynucleotidyl transferase and hybridized to the microarray. Hybridized probes were captured by streptavidin-phycoerythrin conjugates using Fluidics Station 450 and arrays were scanned using GeneChip® Scanner 3000 7G. SNP array data generated in this study have been deposited at GEO with accession number GSE62875. Independent SNP 6.0 data from TCGA colon adenocarcinoma were retrieved from http://​cancergenome.​nih.​gov.

Basic normalisation and segmentation of the microarray data were performed using BioDiscovery Nexus Copy Number 6.0 and the SNP Rank Segmentation algorithm based on Circular Binary Segmentation [15] and default settings. Analyses of absolute allele-specific copy numbers, average ploidy and normal cell content were performed using TAPS [11]. Copy number estimates are included in the Additional file 1.

CNA frequencies (gain to >2 copies per cell, loss to <2 copies per cell, relative gain to >1.25* individual sample average copy number, relative loss to <0.67* individual sample average copy number, homozygous loss, high gain to >3 copies above individual sample average copy number, focal gain and loss of <1 Mb segments, and loss of heterozygosity) and group comparisons were generated using TAPS. Fisher’s exact test was used to estimate statistical significance of observations, generating unadjusted p-values and odds ratios for short segments throughout the genome such that none contained a breakpoint in any sample. A p-value of 0.05 was used as an initial cut-off for significance.

Average copy number or ploidy was calculated as the mean total copy number throughout the autosomes. The difference between the number of autosome arms with 2m0 (two copies with minor allele copy 0, i.e. LOH) or 4m2, and 2m1 or 4m1 was used as a score for evidence of a genome duplication event (using medians of total and minor allele copies throughout each autosome arm, Formula 1).

We used a publicly available set of colon adenocarcinoma samples from The Cancer Genome Atlas (TCGA) [1] to verify which of our findings could be observed in independent data. The TCGA data set differed from the current study in that progression after diagnosis and MSI status was documented only for subsets of the patients. We observed that large CNAs (≥10 Mb) were rare in MSI samples in the current study, affecting less than 5 chromosomes in all but one case, while >93% of MSS cases had CNAs on at least 5 chromosomes (Additional file 1: Figure S2A). TCGA samples with known MSI status showed a similar distribution (Additional file 1: Figure S2B). TCGA samples with unknown MSI status and at least 5 chromosomes affected (n = 37) were considered CIN and included with known MSS samples in order to maximise the number of samples available for validation. MSS/CIN TCGA cases with metastasis at diagnosis (n = 39) were compared with cases without metastasis at diagnosis and with documented survival greater than two years (2-10 years, mean = 5, n = 28). Out of all genomic regions with significantly different frequency of alteration and good effect size (odds ratio ≥4) in our data, only deletion on 1p36 (odds ratio ≈ 6) was independently significant in TCGA. We also observed that deletion or LOH on 18q11.2 (short segment including CABLES1, odds ratio ≈ 3) was associated with dissemination in both data sets, but with a relatively low effect size (Additional file 1).

Subsets (stage II and stage III separately and together, and with postoperative chemotherapy treated cases removed) of the current study displayed associations (odds ratios) very similar to those of stages II-IV combined, supporting that the deletion may be used as a prognostic marker at diagnosis. Peak odds ratios and independent 95% confidence intervals are shown in Table 2. Statistics at base-pair resolution throughout the region are included in Additional file 1. Absolute loss (fewer than 2 copies remaining) and relative loss (fewer than genome average number of copies remaining) were similarly associated with tumour dissemination, though relative loss was more frequently observed (Table 2). For relative loss on 1p36, the difference in frequency between the prognostic groups of the current study (stage II-IV) exceeded 30% on 1p36.13 but was similar throughout 1p36.11-21 (Figure 2A). For the TCGA validation set the difference in frequency exceeded 20% on 1p36.11 and was similar throughout 1p36.11-13 (2B). Total frequency of relative loss displayed very similar profiles in both data sets and peak frequency of loss could be observed near 27 Mb (2C). The difference in frequency between the prognostic groups could be confirmed in the TCGA data set (2B), but a single gene or region responsible for the worse prognosis could not be pinpointed.

In both the current study and the TCGA validation data, 41% of MSS/CIN samples were found to have an average copy number or ploidy above 2.5. Hyperploid genomes may be the effect of whole genome duplications (WGD), which have been implicated in tumours and observed in cancer cell lines [16, 17]. Allele-specific copy number analysis has been used to identify WGD as a frequent event in a variety of cancers including colon cancer, where the frequency of WGD has been estimated to approximately 50% [12]. We investigated evidence of hyperploidy (average copy number >2.5) and WGD in the current study and in the TCGA validation set. For chromosomes present in 4 copies, a WGD event is more likely to have produced 2 copies of each homolog than triplication of one homolog, which is the more likely outcome of successive amplification events leading to 4 copies. Similarly where 2 copies are present, LOH is more likely observed if a WGD event has taken place (after loss of one copy, or followed by loss of two random copies) than if not. We used these assumptions to develop a score sensitive to WGD (see Methods) that would not be directly influenced by the average copy number of the genome. Not surprisingly, average ploidy correlated strongly with WGD score (Figure 3AB). Bimodal distributions of the WGD score for both the current study and the TCGA validation set suggest two groups of samples; one having undergone WGD and the other not. WGD appeared to have occurred in most genomes with hyperploidy and in about one third of all samples. Average ploidy or WGD score did not associate with prognosis or relative loss on 1p36 (p > 0.2, current study MSS, logistic regression). However, absolute loss on 1p36 was strongly associated with, and nearly exclusive to near diploid genomes (p < 10^-5).