Type of tissue for PDL1 readout

Metastasis or primary tumour?

The first studies applied to NSCLC did not show significant difference between the primary and the metastatic sites. Initial recommendations were to utilize what is available. However, a 2020 study made the NSCLC (non-squamous morphology) picture more complex: the following metastatic site (lymph nodes, pleural fluid, soft tissue and adrenal gland but not in those from liver, brain and bone) show higher PDL1 expression. For NSCLC with squamous morphology, higher expression in metastatic sites was not confirmed.

Studies related to Triple Negative Breast Cancers 1, 2 show higher PDL1 positivity in the primary site compared to the metastatic sites. Unfortunately the keynote 355 does not clearly indicates the proportion of primary site vs metastatic site sampled for their study. However since they requested new samples for their study, metastatic sites form probably the majority of the cases. This would suggest that even if the primary sites show higher CPS, the predictive data are based on the metastatic sites. Of note, metastatic samples from the liver consistently show lower CPS compared to other metastatic sites. Also bone metastatic sample especially de-calcified with acid method are associated with high number of false negative.

Biopsy, surgical specimen or cytological sample?

Depends on the underlying algorithm TPS vs CPS. TPS is for the time being only used for the 3D application (Pembrolizumab-NSCLC-22C3 PDL1 assay) and relies on TPS which is the percentage of tumoural cells expressing PDL1. For all other 3D applications (and possibly for all future ones) CPS is performed. CPS is not a percentage but a score which is a fraction from 0 to 100. If the fraction is greater than 100, the score is reported as 100. The numerator includes the number of PDL1 positive immune cells + the number of PDL1 positive tumoural cells; the denominator includes the total number of tumoural cells (positive and negative). In order to be accepted as a positive immune cell, that immune cell has to be in the tumoural bed. Therefore only biopsy and surgical specimens can guarantee the tissue integrity and the relationship of immune cells with the tumoural bed.

Procedure	TPS	CPS
Biopsy	Yes	Yes
Surgical Specimen	Yes	Yes
Esophageal biopsy ultrasound (EBUS)	Yes	No1) Can be used if nothing else available and the number of positive tumoural cells are sufficient to reach a given CPS threshold of positivity. Otherwise one should not report a negative CPS based on cytological or EBUS samples
Cytological sample (FNA or liquid)	Yes2) As long as the cytological sample was fixed in formalin	No3) Can be used if nothing else available and the number of positive tumoural cells are sufficient to reach a given CPS threshold