Interpret 3'/5' Imbalance values

The 3'/5' Imbalance values reporter in Ion Reporter™ Software generates analysis results for Ion AmpliSeq™ gene fusions, such as the Ion AmpliSeq™ RNA Lung Fusion analysis workflow. The imbalance values reporter measures the difference in expression between the 5' assay and the 3' assay of each driver gene. Samples that do not contain a fusion are expected to have similar expression of the 5' assay compared to the 3' assay of the driver gene. Samples that contain a fusion are often expected to have elevated expression of the 3' assay compared to the 5' assay (see the following figure). The 3'/5' Imbalance assay is therefore included for two alternate purposes:

The 3'/5' Imbalance values provide a measurement of the strength of fusion calls that involve ALK, RET, or ROS1 driver genes, whether included or not included in the Ion AmpliSeq™ RNA Fusion panel.

  1. To confirm presence of a fusion from the ALK, RET, or ROS1 driver genes included in the panel, or

  2. If none of the fusions targeted by the panel is detected, to provide evidence of a fusion other than those targeted by the panel but still including ALK, RET, or ROS1 driver genes.

The following approaches interpret the 3'/5' Imbalance values for each gene and are reliable only under the conditions that are described in the following sections:

Most samples that are tested for the 3'/5' Imbalance assay have been lung tumor tissue. Threshold settings for other sample types can be sensitive to varying expression of fusion gene and expression control genes.

We currently do not have sufficient data to determine if the 3'/5' Imbalance value is a predictor for the NTRK1 gene.

Gene

No evidence of a fusion

Uncertain

Strong evidence of a fusion

ALK

≤0.001

0.001—0.015

≥0.015

RET

0.03

0.03—0.55

≥0.55

ROS1

≤0.2

0.2—0.5

≥0.5

For the Oncomine™ Focus Panel, the following table lists the imbalance score thresholds:

Gene

No evidence of a fusion

Uncertain

Strong evidence of a fusion

ALK

≤0.001

0.001—0.0015

≥0.0015

RET

0.03

0.3—0.55

≥0.55

ROS1

≤2.1

2.1—2.1

≥2.1