Copies, alleles, and clones

A tumor DNA result describes a mixture of genomes. To interpret it, separate which sequence version exists, how many copies exist, and which cells carry it.

Sequence versions and parental copies

An allele is a sequence version at a genomic location. Heterozygous means two different versions are present; homozygous means the versions match. A haplotype is a set of nearby variants carried together on a chromosome. Phasing assigns variants to those chromosome copies. NHGRI: allele, haplotype

Germline variants can be inherited and occur broadly across the body's cells. Somatic variants are acquired in a cell lineage. Matched normal means a non-tumor comparison sample from the same person, used to help distinguish acquired tumor changes from their baseline DNA. A research matched-normal analysis and a clinical hereditary-risk test have different validation and reporting scopes.

Copy number and LOH

Copy number and loss of a parental version are different descriptions; neither measures gene function.

Copies of one region, with A and B representing inherited versions. Losing B does not establish that A is nonfunctional.

Copy number is the number of copies of a DNA region. Allele-specific copy number separates the contributions of the two parental chromosome copies. LOH, loss of heterozygosity, means the loss of one of the distinguishable inherited versions. It can arise through deletion or through replacement/duplication that preserves total copy number.

Biallelic inactivation means both functional gene copies are disabled. A mutation plus deletion can support this if the mutation is on the retained copy in the same cancer-cell population. LOH alone does not establish that the retained gene is nonfunctional. NHGRI: LOH, Carter et al.: absolute copy-number analysis

Amplification is an increase in gene/region copy number. An amplicon is an amplified DNA segment that may contain several neighboring genes. Structural variants rearrange larger DNA segments; tandem duplication repeats a segment next to itself. A fusion joins sequences from different genes, but a DNA rearrangement does not guarantee an expressed fusion RNA or protein.

Purity and ploidy

Purity estimates how much of the tested material comes from cancer cells. Specify whether a number refers to cells, DNA, or a model estimate. Ploidy describes genome copy content; tumor analyses often report average copy number across the genome. Whole-genome doubling is a past duplication of the genome. Subsequent gains and losses can obscure that history. Purity and ploidy are fitted together, and more than one plausible fit may explain the data. ABSOLUTE primary study

VAF is different from CCF

VAF, variant allele fraction, is the proportion of usable reads at a site carrying the alternate base. CCF, cancer cell fraction, estimates the proportion of cancer cells carrying the alteration, accounting for purity and copy state. A clone shares acquired DNA changes; a subclone is a branch within that population. “Clonal” is always relative to the sampled tumor and the model.

Hypothetical example: A diploid sample is half cancer cells. Each cancer cell has one mutated copy; normal cells have none. Expected VAF is 25%, although every cancer cell carries the mutation. With a deletion leaving one mutated copy per cancer cell at the same 50% purity, expected VAF is about 33%: 0.5 / (0.5 × 1 + 0.5 × 2). In a simple uniform-copy model, VAF = purity × CCF × mutated copies / (purity × tumor copy number + (1 − purity) × normal copy number). Subclonal copy changes, mapping bias, and uncertainty make real inference harder. Primary tumor-mixture model.

Explain it back

“A 25% VAF means a quarter of the reads carry the variant. It does not mean only a quarter of the cancer cells carry it. We need purity, tumor copy number, and the number of mutated copies to estimate how many cancer cells carry it. Phasing answers a different question: which chromosome copy carries each variant.”

Check yourself: Does “one copy left” mean “no working copy left”? No. The retained copy's function is the next question. Continue to splicing and DNA repair.

Try it

An assay reports a striking value. Which sample, measurement method and comparator do you need before interpreting it?

Answer: Identify the sampled cells, assay definition and reference group before moving from the number to a biological or treatment claim.

Takeaway

The meaning of a measurement depends on its sample, method and comparison.

Sources and scope

General measurement teaching; expert and learner review pending. Sources and definitions are linked beside the relevant methods above.