Use of DNA integrity index in diagnostic testing
Hi readers and welcome back to my blog post on DNA integrity index! Last time we looked at what DNA integrity index is and how it can be measured. Today, we will look at how DII can be used for diagnosing a disease like cancer. So how can a DII ratio help diagnose cancer?
When we say we use DII ratios for diagnosis, we mean that we are testing whether the values of ratios in patients are different from those in healthy people. Most studies say that the cancer patients have longer fragments than shorter fragments. Healthy people tend to have higher amounts of shorter fragments than longer fragments. So, the amount of long or short fragments of cfDNA in a person determines the DII value for that person. Let’s take a study by Pinzani and colleagues as an example.
In 2011, Pinzani and colleagues tried to find a suitable ratio for diagnosing melanoma. They isolated cell-free DNA from the plasma of 79 melanoma patients and 34 healthy people. They measured DNA fragments of four different lengths - 67, 180, 306 and 476 bp. Here, amounts of 67 bp fragments were taken as the amounts of short fragments. The amounts of other three fragment sizes were taken as amounts of long fragments. The idea here was to use three different lengths for long fragments to calculate different DII ratios for each person. Each ratio would then be analyzed to check their ability to differentiate patients from controls.
Using these measurements, they calculated three different DII ratios (180/67, 306/67 and 476/67). As we saw in the previous post, DII is the amount of long DNA fragments divided by the amount of short DNA fragments. If a person has higher amounts of long DNA fragments (for eg. 300 bp) than the short fragments (eg. 67 bp) then this will give a high DII ratio. Otherwise, if a person has lower amounts of long DNA fragments and higher amounts of long DNA fragments, then they will have a low DII ratio. For example, let’s say we measured the concentrations of long (300bp) and short (60bp) DNA fragments in a person X. He has 10 ng/ml of 300 bp fragments and 5 ng/ml of 60 bp fragments. So, the DII for this person will be:
Here the DII is 2. This means that X has higher amounts of fragments of at least 300 bp in length. Now, if we see the inverse of this scenario, let’s suppose X had 5 ng/ml of 300 bp and 10 ng/ml of 60 bp fragments. Then the ratio as per the above equation would be 5 divided by 10, i.e., 0.5. In this scenario, X has higher amounts of DNA fragments below 300 bp in length. So, the DII depends on the quantity of long DNA fragments a person has versus the amounts of short DNA fragments. One point we need to keep in mind here is that the fragments being measured belong to the gene sequence being targeted. So, according to the above example, X has higher amounts of short or long DNA fragments of a particular gene. This is why researchers normally target DNA sequences that are bound to be present in large quantities such as a house-keeping gene, i.e. a gene which is expressed by all types of cells, for DII measurement.
Now, coming back to the study by Pinzani et al, they tested whether these ratios gave different values in patients versus healthy people. They found that the melanoma patients had high values for all the three DII ratios than healthy people. This means that patients had higher amounts of longer DNA fragments in their plasma. These fragments were generally in the size range of 181-307 bp. This means that healthy people tend to have higher amounts of shorter fragments (67-180 bp).
Next, to confirm that these ratios would really be useful as a diagnostic test, they did an analysis called the ROC curve analysis. This website gives a very good explanation of ROC curves. In short, ROC curves or Receiver Operating Characteristic curves are graphical plots that show the ability of a test to discriminate between a disease state and a non-disease state. To create this graph, the true positive rate is plotted against the false positive rate at various cut-off limits of the test. The area under the curve formed by the plot gives a measure of the usefulness of the test in diagnosis.
In this study, the integrity index ratio 180/67 showed the highest specificity and sensitivity for diagnosing melanoma.
Since they found 180/67 to be suitable for melanoma diagnosis, the same group of researchers wondered whether this DII could also help diagnose thyroid cancer. They wondered whether the DII would show a connection with the cytological classification of thyroid cancer. So, in 2017, they tested 97 patients with benign as well as malignant thyroid disease and 49 healthy people. These patients were classified into four groups based on the cytological results – Thy2, Thy3, Thy4 and Thy5. Thy2 group included benign patients. Thy4 and Thy5 groups included patients with suspected or definite diagnosis of malignancy. Thy3 group included patients with indeterminate results or pre-cancerous lesions.
As determined in their previous study, Salvianti et al calculated DII based on the fragment sizes of 180 bp and 67 bp. Patients in all four groups had higher cfDNA concentration, as well as higher DII, than healthy people. Interestingly, the researchers observed that patients in the Thy4 and Thy5 groups had higher DII than those with benign patients. This meant that patients tend to have more fragments with lengths of 180bp or above. But what about the fragments between 67 and 180 bp? The researchers were also curious about this question. To determine the concentration of these fragments, they subtracted the concentration of the 180 bp fragments from that of the 67bp fragments. They found that patients in the Thy2 group tended to have higher amounts of fragments between 67-180 bp. So, DII can be used to differentiate between patients with malignant disease and those with benign disease.
In addition to evaluating DII as a diagnostic tool, the researchers also wanted to see if DII showed any differences after treatment. So, they collected patient samples 3-6 months after surgery. On measuring DII in these samples, they found that the DII had reduced compared to that before surgery.
As in the previous study, here also they checked the ability of DII to differentiate between healthy people, patients with benign disease and patients with malignant disease using ROC curve analysis. They observed that DII could differentiate between malignant and benign disease, as well as between patients with malignant disease and healthy people. Therefore, they concluded that DII ratio 180/67 was suitable to be used as a diagnostic test for thyroid cancer.
So, this was one example of how DII can be used as a diagnostic tool. Various other ratios have been tested by other studies for several cancer types. In general, DNA integrity index has been found to differ between patients and healthy people. Thus, it may be useful as a diagnostic tool for diseases.
If you have any comments or queries, please post them in the Comments section below! Good day!
Excellent.Well written article.
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