Method for Detecting a Genomic Fusion Event

Abstract

The present disclosure relates to methods for detecting and targeting genomic rearrangements, in particular gene fusion events, by targeting a DNA molecule of interest with a set or pool of primers, wherein the forward primers and reverse primers produce a PCR amplification product when a genomic rearrangement is present. The present disclosure also relates to methods of bioinformatic analysis to determine whether or not the detection of an amplification product from the selective PCR is actually indicative of the presence of a gene fusion. The present disclosure also related to related methods of diagnosis and treatment of diseases and conditions associated with such genomic rearrangements, in particular cancers, such as lung cancer.

Description

CROSS-REFERENCING[0001]This application claims the benefit of GB1709675.1, filed on Jun. 16, 2018, which application is incorporated herein in its entirety for all purposes.BACKGROUND[0002]The present disclosure relates to methods for detecting genomic rearrangements, in particular gene fusion events, as well as related methods of diagnosis and treatment of diseases and conditions associated with such genomic rearrangements, in particular cancers, such as lung cancer.[0003]Genetic or chromosomal rearrangements are a type of chromosomal abnormality in which the normal order of the genetic code has been altered. A common genomic rearrangement that is associated with cancer is genetic fusion. A gene fusion event may occur in cancerous or pre-cancerous cells and can be detected in patients to help classify the cancer and determine appropriate treatments.[0004]Existing methods for detecting gene fusions include fluorescence in situ hybridization (FISH), RT-PCR, long range PCR and hybridi...

AI Technical Summary

Benefits of technology

[0012]The present disclosure relates to methods for targeting genomic rearrangement, in particular gene fusion events, by targeting a DNA molecule of interest with a set or pool of primers, wherein the forward primers and reverse primers produce a PCR amplification product when a genomic rearrangement is present. This is achieved by targeting a first region with the forward primers and targeting a second, different, region with the reverse primers. The forward and reverse primers produce an amplification product when they anneal in sufficient proximity to each other. Hence, an amplification product will be produced when a genomic rearrangement has occurred to bring the first and second regions into sufficient proximity. The amplification product is then sequenced to identify the presence and position of the genomic rearrangement. By combining selective amplification and sequence determination it is possible to identify a genomic rearrangement at low allelic fraction even if the PCR produces off-target amplification. The methods disclosed herein do not require a further enrichment step, such as enrichment comprising hybridisation to a probe. The sequence of a reaction product is indicative of the presence of a genomic rearrangement, since the sequence read can be used to directly detect (and characterise) the fusion. Multiple genomic rearrangements can be detected in a single reaction by using multiple sets or pools of primers to detect the genomic rearrangements, wherein each paired set or pool of primers is designed to amplify a different genomic rearrangement (if present). The methods can also be combined with methods to determine the presence or absence of genetic alterations that are not genomic rearrangements, such as single nucleotide polymorphisms (SNPs). This can be achieved by using additional primer pairs that act both as a positive control and to further characterise a disease or disorder or a patient from whom a sample has been taken and analysed. The present disclosure does not require end repair or ligation to enrich for targets of interest and therefore a further advantage is that there is no loss of starting material due to processing prior to fusion detection.

Problems solved by technology

FISH can only be applied to cells or tissue and therefore cannot be used to detect gene fusions in cell free circulating nucleic acids or in DNA already extracted from tissue.

FISH also requires intact nuclei, the need to visually assess individual cells and cannot give the sequence of the breakpoint.

However, this approach can only be applied to mRNA and it is not feasible to identify the breakpoints that have occurred in the genome (DNA). mRNA typically has a short half-life and is therefore a challenging biomarker in heavily degraded samples such as FFPE and circulating RNA (ctRNA).

A significant challenge with this approach is that the fusions will often occur throughout large intronic spaces.

The limitation with these approaches is the requirement for DNA greater than 500 bp in length and therefore they are not suitable for fragmented DNA such as FFPE or cfDNA.

These long-range PCR methodologies are also not compatible with Next Generation Sequencing Technologies due to the short-read length (<500 bp) of most next generation sequencing platforms without further complex steps such as fragmenting the DNA then ligating on adaptors.

This methodology also requires a potentially large number of individual PCR reactions to assess each sample or complex steps such as positive selection using biotin-labelled primers in order to multiplex such a reaction.

As these steps are highly inefficient ˜70% of starting material is lost prior to hybridisation, limiting the ability to detect fusions present at low allelic frequencies.

Finally, this approach is time consuming, normally requiring overnight incubation of target and probe to enable hybridisation.

However, this approach is time consuming and requires the ligation of universal adapters to DNA ends.

The inefficiency of ligation limits the ability to detect fusions present at low allelic frequencies.

It also increases complexity and cost of the workflow as an additional hybridisation probe is required.

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