Question: Why is normal blood used for matched tumor (instead of adjacent norm tissue)?
4
gravatar for umn_bist
3.3 years ago by
umn_bist320
umn_bist320 wrote:

I am using RNA-seq for somatic variant calling and but was curious why it is standard practice to use peripheral blood as normals. Wouldn't the expression profile of blood be of multiple cell types that could be different from the tumor cell type? I feel like using adjacent normal tissue would be a stronger normal to match with but it seems customary to use blood instead. Would love to hear your thoughts.

rna-seq • 2.8k views
ADD COMMENTlink modified 3.3 years ago by Chris Miller21k • written 3.3 years ago by umn_bist320
9
gravatar for Chris Miller
3.3 years ago by
Chris Miller21k
Washington University in St. Louis, MO
Chris Miller21k wrote:

Blood is not typically used as a normal for expression differences. In fact, the most common paradigm is tumor/normal DNA and tumor only RNA. As you imply, expression or methylation profiles will be quite different between RNA from a solid tumor and RNA from the blood.

You can use matched normal blood with tumor RNA for variant calling, as the background mutational profile of SNPs will be preserved in both. The caveat is that you're going to miss mutations in genes that are poorly expressed (which may or may not be fine, depending on your experimental design)

It's also worth noting that adjacent normal tissue is often a poor control anyway, for two reasons: 1) tumor infiltration (or premalignant states) is often observed in nearby tissue, meaning that causative mutations/expression changes will be present in your normal (which is bad). 2) changes in the microenvironment may cause quite different expression profiles than you'd see in a true normal.

That all aside, you often can't collect matched normal tissue - can't just go scooping out a chunk of healthy brain to match up with your GBM tumor. Even if you could collect truly matched normal tissue (from the other breast in BRC, for example), there's the issue of subjecting the patient to another procedure that carries risks and may not provide clear benefit.

ADD COMMENTlink modified 3.3 years ago • written 3.3 years ago by Chris Miller21k

so, how to get the control, thanks a lot.

ADD REPLYlink written 7 months ago by linouhao0
4
gravatar for Manuel Landesfeind
3.3 years ago by
Göttingen, Germany
Manuel Landesfeind1.2k wrote:

Like dariober pointed out, blood is easier to obtain. In fact, if you want to collect a "normal" sample of the exact same tissue, you have to extract material at a location that is distinct to the tumor to make sure that it does not contain any tumor material. This would mean to further destroy tissue of an already damaged organ which is probably not such a good idea.

Regarding differences, if you want, for example, identify the mutations causing the tumor, it is not required to have a sample from the exact same tissue. You have to analyze both samples and ignore all variants found in the normal sample, i.e., mutations that are inherent to the patient and found in all tissues including the blood.

ADD COMMENTlink modified 3.3 years ago • written 3.3 years ago by Manuel Landesfeind1.2k
1

I am using RNA-seq for somatic variant calling

I think the OP has a more specific concern: how to account for varying expression of transcripts in different tissues when using the cDNA library for somatic variant calling. I'm not sure how to answer this except to require that remove regions that are lowly expressed in either tissue and then call only those regions with sufficient coverage in both.

ADD REPLYlink written 3.3 years ago by Matt Shirley9.0k
2
gravatar for dariober
3.3 years ago by
dariober10k
WCIP | Glasgow | UK
dariober10k wrote:

I think part of the reason is that blood is easier to obtain.

ADD COMMENTlink written 3.3 years ago by dariober10k

But wouldn't the profile of blood and tumor tissue be very different? Is it similar enough to disregard the potential false positives that arise from the difference? Thank you for your input.

ADD REPLYlink written 3.3 years ago by umn_bist320
1

Yes, but then the main problem is you need justification to collect those tissue. Sometimes, we can only compromise...

ADD REPLYlink written 3.3 years ago by Sam2.3k
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