Neoantigens with RNA expression = 0 tiered as Pass

[biounix]

Hi,

I found that some of my variants in the aggregated results table are tiered as Pass despite their RNA Expr values being 0. Since we have RNA expression but not RNA VAF, I assumed that the Expression Criteria filter was disabled, as I understand from here and here. However, I still see some neoantigens with an expression value of 0 classified as NoExpr, which suggests that the Expression Criteria filter is active.

Could you help me understand what’s happening?

Thanks a lot!

[susannasiebert]

Would you be able to share a VCF file and an aggregate report file that each contain the two variants in question? This would help us in investigating this issue further.

On the top of my head, there is a difference between a true 0 as in there was a RNA expression entry with a value of 0 and "NA" where there was no RNA expression entry or where RNA expression estimation was not run. The latter is meant in the issue you referenced, which would still be binned into the Pass tier if all other criteria are met. That doesn't sound like what is happening in your case though since you said that both have a value of 0 so I would like to debug this further.

[biounix]

Sure, @susannasiebert! Please, find the files here. You can spot a couple of entries with an expression value of 0 in the Pass tier, a few more in the Subclonal category, and about a dozen in the NoExpr tier.

Thanks for your help!

[susannasiebert]

Ok, so I dug into this some more and the difference is that the for the variants in the Pass tier the candidate passed all of the other criteria, including the binding cutoff. For the ones in Subclonal tier it only failed the subcloncal assessment. In both cases it considered the allele expression criteria a pass because of the RNA VAF being NA. The ones in the NoExpr tier either failed the binding assessment, the TSL assessment, or more than one other criteria. It's pretty broad because it's one of the last tiers we evaluate if it wasn't able to be tiered into any of the other ones and the only criteria we check at that point is whether the gene expression or rna vaf if 0. We have some documentation, in case that might help clear things up a bit.

We've had a few confusions with this approach and have an open issue to make some updates to the tiering. I will make sure that this issue is reevaluated at the same time.

[biounix]

Thank you very much for the explanation and for clarifying the behavior.

Without RNA VAF, I thought the Expression Criteria would be completely disabled and no variants would be classified as NoExpr at all.

Thanks again for digging into this.

[susannasiebert]

You're welcome. Is it ok if I close this issue? You are welcome to subscribe to issue #1188 to keep track of the improvements I described.

[biounix]

Sure!

Thanks again.