The Yale Study

Dr Pusztai and colleagues used paired formalin-fixed paraffin-embedded tissue blocks of primary and metastasis from the Yale Pathology Tissue Services. Two independent cohorts were used and TILs and PD-L1 expression were assessed in both.

For cohort 1, the researchers obtained full sections of paired metastatic and primary tumors of 45 patients. For cohort 2, the researchers assembled tissue microarrays (TMAs) from 55 other patients, which included 42 paired primary and metastatic lesions.

Stromal TIL was determined from hematoxylin eosin staining of tissue samples. PD-L1 was determined from an immunohistochemical analysis using a rabbit monoclonal antibody. The expression of 730 immune-related genes and 40 housekeeping genes was assessed using Nanostring PanCancer Immune Profiling assay.

The immune genes were grouped into 14 immune cell metagenes (total T cells, Th1, Treg, total CD8, exhausted CD8, cytotoxic T, B, NK, NK-CD56, mastoid cells, CD45, dendritic cells, macrophages, and neutrophil) and 22 immune functions. Metagene scores were calculated as the geometric mean expression of member genes, and scores were also calculated for previously identified (via previously published studies) prognostic metagenes and metagenes thought to predict response to immunotherapies.

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Median time between obtaining the primary tissue sample and matching metastatic biopsy was 3.4 years and patients received a median of 1 prior line of therapy for metastatic cancer before biopsy of the metastatic sample.

TILs and PD-L1 Levels

In samples from both cohorts of patients, TILs and PD-L1 expression was lower in metastatic samples compared with their paired primary tumor samples. PD-L1 staining was mostly localized to the stromal immune cells, rather than tumor cells.

PD-L1 positivity was seen in significantly more primary tumor samples in cohort 1 (52% vs 14% for metastatic samples; P = 0.0004) as well as in cohort 2 (22% vs 7% for metastatic samples; P = 0.03). Expression levels of mRNA for PD-L1 were also significantly lower in metastatic lesions.