1. Techniques that can be used to detect 1p/19q codeletion.

Technique	Brief description
FISH	FISH testing uses fluorescently labelled probes that are designed to hybridise to specific chromosomal locations. It can be performed on FFPE, and on fresh or frozen tissue. In this technique tissue architecture is preserved. To test for chromosome 1p/19q codeletion, chromosomes 1 and 19 are analysed on separate slides. FISH probes corresponding to regions of 1p or 19q labelled using 1 colour, and control probes on 1q or 19p labelled in another colour (as 1q and 19p seem to remain unaffected) are used. Many commercially available probes hybridise to loci at 1p36 and 19q13, although the FISH probes used at different centres may not target exactly the same loci (Pinkham 2015). Normal nuclei show a diploid signal ratio of 2/2 (2 signals from 1p or 19q and 2 signals from 1q or 19p). Absolute deletions will theoretically result in 1 signal from 1p or 19q in the presence of 2 signals from the control loci. There is no consensus on cut‐offs to diagnose codeletion. This is demonstrated by the fact that the EORTC study 26951 and the RTOG study 9402 used slightly different criteria (Pinkham 2015). Some laboratories define cut‐offs based on the percentage of cells with deleted and imbalanced signals, some define cut‐offs based on ratios calculated by dividing the total number of test probes by the total number of control probes, and some combine percentage and ratio cut‐offs.
CISH	This is a very similar technique to FISH, but instead of using fluorescent labelling, the probes are labelled with a marker such as biotin, digoxigenin or dinitrophenyl, and then this marker is detected using antibodies or streptavidin (that binds biotin) that is conjugated to enzymes such as horseradish peroxidase or alkaline phosphatase. The presence of the probe can then be visualised in the presence of a substrate that undergoes a colour change in the presence of the enzyme. The advantages of CISH is that it does not require a fluorescence microscope and staining is permanent.
PCR‐based LOH assays	This technique analyses polymorphic microsatellites that are dispersed throughout the genome. Different alleles have different numbers of repeats. PCR amplification of regions containing polymorphic microsatellites can therefore result in different length PCR products. If an individual is heterozygous (has 2 different alleles) for a microsatellite, PCR of this region will result in 2 different length products. If heterozygosity is lost, only 1 length product will be obtained. An individual must be heterozygous for a microsatellite for it to be informative, and DNA from normal tissue is required to determine this. LOH can be determined by comparing the ratio of PCR products of different lengths obtained from normal and tumour tissue. Primers that amplify regions containing microsatellites on 1p and 19q can be used to determine whether 1p and 19q are codeleted. However, there is no consensus on location or number of microsatellites analysed.
RFLP analysis	LOH can also be detected using RFLP analysis. In RFLP, restriction enzymes that recognise specific sequences are used to cut DNA, resulting in fragments of specific sizes. Different alleles may contain cut sites, or the DNA fragment that the restriction enzyme produces after digestion may be expected to differ due to different numbers of repeats in different alleles. Therefore, in a similar manner to PCR, LOH can be detected through loss of fragments of a specific size from informative loci (where an individual is heterozygous in normal tissue).
Comparative quantitative PCR	Comparative quantitative PCR compares the amount of PCR product obtained from 1p/19q with PCR product obtained from other chromosomal regions. If a deletion is present, less PCR product will be obtained. This technique has the advantages that heterozygosity at loci is not required, neither is a sample of normal tissue.
MLPA	MLPA uses probes designed to hybridise to specific regions of the genome that have been split into 2. Each probe 'half' also contains sequences corresponding to universal forward and reverse binding sites for PCR primers, and 1 'half' contains a region of varying length to help identify the probe later. The primers are hybridised to denatured sample DNA (e.g. from a tumour). The next step is ligation. Only probe halves that are hybridised to adjacent sequences on the sample DNA will be ligated together. PCR, using primers corresponding to the universal binding sides contained in the probes, is used to amplify the probes. Only those probe halves that were ligated together will be amplified to any extent, as it is only these products that contain the binding sites for both the forward and reverse PCR primers. The PCR products can then be separated by length, and quantified. The results are then normalised internally (by comparing reference probes with target probes), and then compared with reference samples. Heterozygous deletions can be identified as a probe ratio of 0.5 will be observed, and heterozygous duplications from a probe ratio of 1.5. Usually, probe ratios < 0.7 or > 1.3 are regarded as indicative of a heterozygous deletion (copy number change from 2 to 1 allele) or duplication (copy number change from 2 to 3 alleles), respectively (Eijk‐Van Os 2011).
CGH	In CGH, differentially labelled genomes from the tumour (the test genome) and normal tissue (the control genome, which does not need to be from the same person) are simultaneously hybridised to normal metaphase chromosomes. Changes in copy number, caused for example by loss or gain of regions, will alter the ratio of the 2 genomes. If 2 different fluorochromes are used to mark the genomes (or detect the labels), changes in copy number can be revealed from the relative intensities of fluorochromes used to detect the 2 genomes. CGH detects DNA sequence copy number changes relative to the mean copy number in the entire tumour sample. However, signals can be normalised using the sex chromosomes, which may help if a tumour is known to be normal for these chromosomes.
aCGH	aCGH follow the same principles as CGH, but instead of the 2 genomes being competitively hybridised to metaphase chromosomes, they are hybridised to a microarray. The theoretical resolution of aCGH is greater than that of traditional CGH.
SNP arrays	An SNP array is a type of DNA microarray. SNP arrays allow both copy number status and genotype to be determined, allowing detection of both losses and copy‐neutral LOH. SNPs are variations at a single position in a DNA sequence. Since individuals usually inherit 1 copy of each SNP position from each parent, the individual's genotype at a SNP site is typically either AA, AB or BB. To detect abnormalities using SNP arrays, sample DNA is fragmented, labelled and hybridised to an array containing immobilised allele‐specific oligonucleotide probes (1 probe for each allele). The signal intensity associated with each probe is then measured. Copy number changes can be detected from the intensity of signal. By comparing the result for each SNP with those from normal tissue, or by using a hidden Markov model, LOH can be detected. In the rare case of 2:2 tetraploidy, it is possible that SNP arrays will not be able to distinguish absolute from relative deletions.
Methylation arrays	Genome‐wide DNA methylation array data can also be used to detect 1p/19q status, as reported in Capper 2018b. In methylation arrays, specific regions of the genome that may be modified by methylation are investigated. The array has 2 probes for each region, 1 for the methylated and 1 for the unmethylated. To detect copy number variations, the signal from both probes (the methylated and unmethylated) for a specific region are added together and compared with a reference genome.
NGS	NGS refers to post‐Sanger sequencing technologies including sequencing‐by‐synthesis, sequencing‐by‐ligation and ion semiconductor sequencing. While traditional Sanger sequencing sequences a single DNA sequence, NGS is capable of sequencing multiple sequences simultaneously. Techniques have been developed to detect LOH and copy number variations using NGS. Deletions can be detected by relative perturbations in the read depth. LOH can be detected when the ratio of alleles at a heterozygous SNP site is perturbed.

aCGH: array comparative genomic hybridisation; CGH: comparative genomic hybridisation; CISH: chromogenic in situ hybridisation; DNA: deoxyribonucleic acid; EORTC: European Organisation for Research and Treatment of Cancer; FFPE: formalin‐fixed, paraffin‐embedded tissue; FISH: fluorescence in situ hybridisation; LOH: loss of heterozygosity; MLPA: multiplex‐ligation‐dependent probe amplification; NGS: next‐generation sequencing; PCR: polymerase chain reaction; RFLP: restriction fragment length polymorphism; RTOG: Radiation Therapy Oncology Group; SNP: single nucleotide polymorphism.