Chung-Chee (Jeff) Chang, MD, PhD
A Pathology Perspective on Biomarker Testing in NSCLC
So, in general, the more of the tissue, the better for biomarker testing. In general, 1 cm3 volume of tissue is appropriate for the biomarker testing. For most of the resected tumors, this is usually not a big problem. However, nowadays, most of the specimen that we’ve got is actually from needle core biopsy or from the fine needle aspiration. Usually, this type of procedures do not produce that much amount of tissue. Therefore, most of the laboratories nowadays, they will try to extract the DNA, no matter how much amount of tissue they can get. And then, they can decide if the DNA’s enough or not.
The amount of DNA or the quality of the DNA actually depends on how many cells are in the tissue and how the tissue has been fixed. And a lot of different factors can impact the amount of DNA that can be recovered from the tissue. And if they’ve got enough DNA, then they will go ahead and run the test anyway.
In the 2013, the College of American Pathologists, the guideline indicates only formalin-fixed paraffin-embedded tissue is a good material. So at that time that we followed the guideline, we’d only accept these formalin-fixed paraffin-embedded tissue blocks. However, in the 2018 guidelines, they extended as appropriate specimen type to cytology specimens, as well, such as smears or the cell block from the fine needle aspiration. And so since then, we have been also accepting this type of the material.
Besides the volume of the tissue specimen, another important factor is actually the percentage of the cancer cells within the tissue. Generally, we need about 20% of the cancer cells to be in the slides or the tissue to be able to detect all the mutations. Therefore, it will be important to get the amount of that percentage of cancer cell in the specimen. And this is particularly important for the interventional radiologist, because when they try to take the specimen, there is no way by their naked eye to know how many percent of the cancer cell actually is within the specimen.
And so there’s a couple ways to overcome this problem. One way is to increase the size of the needle they use to get the tissue. But this potentially could increase the complication rate associated with the procedure. And so another good way to do this is actually to have a cytotechnologist or a pathologist actually to do an onsite evaluation to see how many cancer cells are in the specimen. This way, you can ensure there’s enough cancer cells in the specimen that was obtained.
So the current CAP guideline actually requires a lab to complete all the molecular testing within 10 days the lab receives the specimen. This is because that 10 working days, within 10 working days is really important for the oncologist and the patient to get the optimal treatment decision information.
For our own institution, we actually established a protocol. So we actually test older lung cancer patients regardless of their clinical stage, even in the guideline they suggest to do just the advanced-stage patient. But this way we actually eliminate the needs for the oncologist to call us, and so this actually shortens the turnaround time. However, in some institutions, if they don’t have such a policy to test older lung cancer patients, then it’s important for the oncologist to call up the lab, to perform biomarker testing on the patients that are in the advanced stage, for the appropriate test. And this will be important to shorten the turnaround time, to make sure the test can be done within 10 working days after the patient have a procedure.
For the pathologist, the most important job is actually to select the appropriate tissue blocks or the slides for the biomarker testing. As indicated, 20% of the cancer cells in the slide is important for the test. And so if the overall percentage of cancer cells within a slide is less than 20%, the pathologist needs to actually look under the microscope, using their marking pen to select the area that has more than 20% of the cancer cells. The lab can then macrodissect out the particular area is enriched in cancer cells to do biomarker testing. And also, if for any reason the lab are submitting some block that can continuously fail the biomarker testing, then the pathologist needs to go back and pay attention to their formalin solution to make sure the intended, preferred pH is actually accurate, and also, the fixation time of their specimen is appropriate. For example, for the small core biopsy specimen, it probably needs to be fixed about 6 to 12 hours, and for a big specimen, then it’s about 12 to 18 hours.
So this guideline has been very helpful to us in terms of select appropriate specimen type for testing. For example, in the 2013 guidelines, they recommend just using formalin-fixed paraffin-embedded tissue blocks, and so that has been the way we’re doing it from 2013 to 2018. But this year, in 2018, the new guideline actually indicated that the cytology specimen, such as smears or the cell block from the fine needle aspirate cytology specimens, is also appropriate type of the sample. And so we actually adopted that guideline after we do some brief in-house validation to ensure those type of specimen is adequate. And also, because the new guideline actually added a lot of optional markers for testing, and that including like HER2/neu mutation, MET exon 14 skip, and because these additional markers let us decide to actually change our current single-chain assay to a multiple-chain next-generation sequencing-based assay, so we can test all the different types of driver mutation, probably total, maybe about fifty something genes.
I would like to share two processes that we established over the last 5 years that has been tried to maximize the biomarker testing efficacy for the lung cancer patients.
First, we established a cascade testing system. As mentioned, we routinely test older lung cancer patients for the biomarkers. This actually helped to eliminate the need for the oncologist to call us. And then this shortened the turnaround time for the patients. However, when we do this, we also increase the possibility of testing some patient who may not really need the testing at this point. And so to avoid unneeded tests, we actually also did this cascade type of testing. So for older lung cancer patients, we test the KRAS mutation first, because the KRAS mutation is the most common one, occurring in about 30% to 40% of lung cancer patients. And if a patient has a KRAS mutation, they usually don’t have other type of driver mutation. And so we test the KRAS mutation first. By doing so, if they already mutated, then they don’t need further testing, and so this will exclude about 30% to 40% of patients, need additional test. If the patient does not have a KRAS mutation, then we move forward to test additional driver mutation that’s recommended by the guideline.
And another thing we establish is, we now routinely precut all the core biopsy for like 30 consecutive unstained slides. This is because we find out when we try to recut the specimen for molecular marker testing, after the initial morphological evaluation, tends to lose in a lot of tissue. And so now we cut 30 unstained slides. We stain first, eighth and the 15th slide to make sure we have enough tumor cells in the slides. And then we can then use in the first to the seventh unstained slides to do like immunohistochemical stains, such as PD-L1 or the other diagnostical markers needed for a diagnosis. And then the eight to 14th slides can then be used for biomarker testing. By doing so, we have a very high success rate of testing all of our patients for biomarkers.
CASE STUDY ON BIOMARKER TESTING IN ADVANCED NSCLC
Patient Case: Biomarker Testing in Advanced NSCLC