TRK3 copy number, what does it mean?

Predicting mRNA expression from copy number:  It’s not working!

  1. Total TRK3 copy number is a weak predictor of the copies of the “minor allele” but not a predictor of the mRNA transcripts.
  2. Amplified TRK3 gene copies do not take up the same amount of space in base pairs on chromosome 15.
  3. On average, some TRK3 genes take up less space than the wild type gene suggesting incomplete amplification.
  4. Promoter methylation may be another factor to examine.

Can the copy number be used to predict the overall expression as measured by the mRNA Z-score?

It should be noted that mRNA data was not available for each and every patient in the the COSMIC database. As one would expect, there is a weak correlation between the total copy number and that of the minor alleles (Fig 1A). An R = 1 value would mean an exact 1:1 correlation. R = 0.53 suggests that the canonical and minor alleles might be differential duplicated from one cancer to the next. The copy number is absolutely not a predictor of Trk mRNA transcripts. R = 0.06 is very close to R = 0, absolutely no correlation whatsoever.

graph showing minor alleles on y axis and copy number of TRK3 gene on x axis. second graph showing mRNA expression on y axis and copy number of x axis

Figure 1. TRK3 total copy number can A.  predict the copies of minor alleles, but cannot B. predict mRNA expression.

 

How much space, in base pairs, is occupied by duplicated genes?

The COSMIC database listed information for 45  TRK3 gene amplification tumors. The wild type position for the TRK3 gene is marked by an arrow at approximately position 49 on the y-axis of the plot (Figure 2). Tumor 44 was listed as spanning base pairs 19,811,075 to 98,827,342 on chromosome 15. This particular tumor was used in calculations but is not shown on the graph in Fig 2.  An arrow marks the span of TRK3 gene copies in tumor #1 at the bottom of the plot in Figure 2.  Note the distance between the solid and empty squares at position 49 (wild type) and position 1 (tumor 1).

 

Distance base pair map of TRK gene(s) on chromosome 15 taken from COSMIC database comparing wild type with tumor TRK genes

Figure 2 Distance (in base pairs) map of the TRK gene(s) on chromosome 15, COSMIC database. The arrow at the top marks the region of chromosome 15 occupied by the wild type, single copy of TRK3.

 

Even when the TRK3 gene is amplified, the position that it/they occupy may be more confined than in the wild type gene as seen by the intersection of the filled and hollow orange squares.

Can estimating the space occupied by each TRK3 gene tell us anything?

The COSMIC database gives regions of chromosome 15 where copies of the TRK3 gene reside. COSMIC also tells us the average ploidy. Let’s assume a few things just for the sake of presenting the data and thinking about it.

  1. Just one copy of chromosome 15 gets duplicated out of control. The other chromosome 15 remains at one copy per cell. For the sake of examining the data, let the total copies of chromosome 15 be the average ploidy minus one.
  2. Just for viewing the data, let us assume that one chromosome 15 contains one largely normal copy of the TRK3 gene. All the other copies of TRK3 reside on the one or more other copies of chromosome 15. If the average ploidy is 5, there is one normal chromosome 15 and 4 altered chromosome 15s.
  3. The abnormal chromosome 15s contain the same number of aberrant copies of the NTRK3 gene. If the copy number is 13, there is one copy of NTRK3 on the normal chromocome 15 and an average of 12 copies occupying the 4 altered chromosome 15s.
Cartoon showing normal copy of TRK3 gene with amplified chromosome 15

Figure 3. Assumptions for examining data.

Another way to view these data is to calculate the distance occupied by the TRK3 genes based on the starting and finishing position on chromosome 15 given by COSMIC (Figure 4A).  A histogram of the estimated distance occupied by each copy of TRK3 (Figure 4B) suggests that adjacent regions get amplified in some tumors and only portions of TRK3 get amplified in other tumors. The blue arrow marks the expected distance of the wild type TRK3 gene.  Note that the scale in Panel 3A (in tens of millions of base pairs ) is over 10x that of panel 4B (in millions of base pairs).

Frquency histograms of TRK3 gene distance in COSMIC database

Figure 4 Frequency histograms of TRK3 gene distance A. as given for total copies in the COSMIC database B. estimated average distance based on ploidy and copy number also given in the COSMIC database.

 

Some evidence of amplification of only part of TRK3

A closer examination of some of COSMIC’s data for the TRK3 gene reveals some interesting things to think about (Fig 5).  Going from the top row to the bottom:

  • A diagram at the top describes TrkC protein domains.
  • No complex mutations or deletions to report.
  • All of the deletions are frame shift mutations.
  • The rose colored bars are CNV by domain.  CNV ranges from 39 to 41.  The five regions of TRK3 are not being copied equally.
  • While some TrkC mRNA over expression was seen, in most cases it occurred without TRK3 gene amplification
  • The number of cases of under expression as measured by mRNA Z-score was the same as the cases with hyper methylation of the TRK3 promoter.

 

Cartoon showing TRKC domains with frame shift mutations

Figure 5. Some additional things to think about regarding TRK3 in cancer.

 

Gene amplification would appear to be far from the entire story.

Amplification of the products of gene rearrangements?

It is interesting to note that the ETV6-TRK3 gene rearrangement may result in copy number gains for the ETV6 and TRK3 genes and loses for genes centromeric to ETV6 and telomeric to NRK3.

Lambros MB, Tan DS, Jones RL, Vatcheva R, Savage K, Tamber N, Fenwick K, Mackay A, Ashworth A, Reis-Filho JS.(2009) Genomic profile of a secretory breast cancer with an ETV6-TRK3 duplication. J Clin Pathol. 62(7):604-12. PubMed

A simple test to detect TrkC over expression

A simple test exists to detect TrkC over expression in tumors no matter what it is fused to. In this simple test to determine eligibility or a clinical trial testing a specific Trk inhibitor the following methodology is used

  1. The tumor is subjected to immunohistochemistry using an antibody that recognizes many receptor tyrosine kinases (RTK) that are known to drive cancer
  2. A second section of the tumor is stained with antibody against the three RTK that the small molecule being tested inhibits: ALK, ROS1, and the three isoforms of Trk.
  3. If one of these three RTK (protein!) is detected, the mRNA is extracted, amplified using the appropriate primers, and subjected to next generation sequencing.

This method is blind to how many times a gene has been amplified. At the end of the day, maybe all we care about is if there is an out of control kinase like Trk C driving a cancer.  And if a small molecule can stop it!