This method using the 22C3 antibody concentrate to determine PD-L1 expression in both tumor tissue and cytology specimen will expand the ability of laboratories to assess selection of patients with non-small cell lung cancer for immunotherapy in a reliable and reproducible manner. The main advantage of this technique is that it's highly concordant with the gold standard assay and will support reliable, high-quality PD-L1 testing across regions worldwide. In general, pathologists new to this method will struggle because of the granular staining occasionally observed.
Moreover, the staining of the human cells is somewhat more intense than that observed with the gold standard. Therefore training of pathologists is necessary to obtain correct interpretation of PD-L1 staining with the 22C3 LDT. Demonstrating the procedure will be Mrs.
Marame Hamila, a technician from my lab. To begin, fix the tumor tissue in 10%neutral-buffered formalin in a cassette and embed the cassette in paraffin as outlined in the text protocol. Embed the infiltrated tissue inside a mold that is filled with molten paraffin.
Let the tissue stay in the mold until the paraffin has solidified. After this, use a microtome to section the paraffin-embedded tissue at a thickness of three micrometers. Transfer the paraffin ribbon to a positively-charged glass microscope slide.
Then, dry the slide for one hour at 37 degrees Celsius. First, collect the bronchial washings in a preservative solution. Transfer the washings to a 50-milliliter conical tube.
Add two grams of DL-Dithiothreitol and vortex the tube for 30 minutes, then centrifuge at 250 times g for five minutes at room temperature. Remove the supernatant and add 10 milliliters of a mucolytic solution. Shake the sample at medium speed for 20 minutes and centrifuge at 250 times g for five minutes at room temperature.
After this, remove the supernatant and deposit the cell pellet into a collection tube containing 10%neutral-buffered formalin. Add four drops of a cell block preparation agent. Transfer the cell pellet to a cassette.
Fix the cell pellet for paraffin embedding and sectioning using the process previously described for preparing tumor tissue samples. To begin, power on the autostainer and the computer. Double click on the autostainer icon and choose the user.
Click on Create Label and then on Protocol. Double click on the 22C3 protocol. Fill in the patient's ID and click Print.
After this, stick the label onto the slide. Press the button on the chosen slide drawer to open it and place the labeled slide on the thermal pad with the label facing upwards and inwards. Close the slide drawer.
Next, remove the caps from the dispensers. Load the reagents on the reagents rack. Open the hood of the stainer and place the reagent racks on the reagents carousel, making sure that they fit properly and hold in position, then close the hood.
In the software, click on the instrument that will be used and then click Running. At the end of the IHC procedure, use tap water with a single drop of cleaning solution to rinse the slide for several seconds. Dehydrate the slide by immersing it sequentially in two ethanol baths for several seconds each, then place the slide into a coverslipping machine to automatically load the cover slip.
To begin assessing the quality of the PD-L1 staining, analyze the positive and negative controls before examining the patient specimen. Using a microscope, confirm the presence of at least 100 viable tumor cells. At a low magnification of 4 times, evaluate all well-preserved positive and negative tumor areas.
After this, score partial or complete cell membrane staining and calculate the tumor proportion score by assessing the proportion of PD-L1-positive tumor cells relative to all tumor cells present in the well-preserved tumor areas. In this study, the tumor proportion score or TPS is evaluated for paired-tissue biopsy samples in cells blocks prepared from bronchial washes or pleural effusions. Representative staining patterns with biopsy specimens are used to compare the 22C3-antibody concentrate against the PD-L1 IHC 22C3 companion assay.
The intraclass correlation coefficient, which is used to measure the correlation of the TPS score as a continuous variable, is 99%between the LDT and the gold standard. Representative staining patterns with cytology specimens are also used to compare the 22C3-antibody concentrate against the PD-L1 IHC 22C3 companion assay. The concordance rate of biopsy and cytology samples with the LDT is greater than 95%when using either of the TPS cut points.
The intraclass correlation coefficient while using TPS as a continuous variable is between 0.88 and 0.90. Once mastered, this technique gives a high concordance rate with the gold standard in evaluate the cutoff for positivity or tumor histology for both tissue and cytology specimens. While attempting this procedure, it's important to remember that the samples must be evaluated with reference to the HE slide and in some difficult cases complementary stains to assess immune cells may be performed to exclude misinterpretation of the PD-L1 expression in tumor cells only.
Following this procedure, other specimens like fine-needle biopsies, EUS-FNAs or bronchial brushes can be evaluated. After its setting in the clinics, the assay robustness needs to be maintained over time by seeking for accreditation, by following standard operating procedures, and by regularly participating in external quality assessment schemes. After watching this video, you should have a good understanding of how to implement the optimized protocol using the 22C3 anti-PD-L1 antibody concentrate on a widely available IHC autostainer both biopsy and cytology samples from patients with non-small cell lung cancer.
