The Expression and Prognostic Impact of Immune Cytolytic Activity-Related Markers in Human Malignancies: A Comprehensive Meta-analysis

Abstract

Background: Recently, immune-checkpoint blockade has shown striking clinical results in different cancer patients. However, a significant inter-individual and inter-tumor variability exists among different cancers. The expression of the toxins granzyme A (GZMA) and perforin 1 (PRF1), secreted by effector cytotoxic T cells and natural killer (NK) cells, were recently used as a denominator of the intratumoral immune cytolytic activity (CYT). These levels are significantly elevated upon CD8+ T-cell activation as well as during a productive clinical response against immune-checkpoint blockade therapies. Still, it is not completely understood how different tumors induce and adapt to immune responses.

Methods: Here, we calculated the CYT across different cancer types and focused on differences between primary and metastatic tumors. Using data from 10,355, primary tumor resection samples and 2,787 normal samples that we extracted from The Cancer Genome Atlas and Genotype-Tissue Expression project databases, we screened the variation of CYT across 32 different cancer types and 28 different normal tissue types. We correlated the cytolytic levels in each cancer type with the corresponding patient group's overall survival, the expression of several immune-checkpoint molecules, as well as with the load of tumor-infiltrating lymphocytes (TILs), and tumor-associated neutrophils (TANs) in these tumors.

Results: We found diverse levels of CYT across different cancer types, with highest levels in kidney, lung, and cervical cancers, and lowest levels in glioma, adrenocortical carcinoma (ACC), and uveal melanoma. GZMA protein was either lowly expressed or absent in at least half of these tumors; whereas PRF1 protein was not detected in almost any of the different tumor types, analyzing tissue microarrays from 20 different tumor types. CYT was significantly higher in metastatic skin melanoma and correlated significantly to the TIL load. In TCGA-ACC, skin melanoma, and bladder cancer, CYT was associated with an improved patient outcome and high levels of both GZMA and PRF1 synergistically affected patient survival in these cancers. In bladder, breast, colon, esophageal, kidney, ovarian, pancreatic, testicular, and thyroid cancers, high CYT was accompanied by upregulation of at least one immune-checkpoint molecule, indicating that similar to melanoma and prostate cancer, immune responses in cytolytic-high tumors elicit immune suppression in the tumor microenvironment.

Conclusion: Overall, our data highlight the existence of diverse levels of CYT across different cancer types and suggest that along with the existence of complicated associations among various tumor-infiltrated immune cells, it is capable to promote or inhibit the establishment of a permissive tumor microenvironment, depending on the cancer type. High levels of immunosuppression seem to exist in several tumor types.

Keywords: cancer immunotherapy; granzyme A; immune cytolytic activity; metastasis; perforin 1; survival rate; tumor-associated neutrophils; tumor-infiltrating lymphocytes.

Figure 1

(A) Varied immune cytolytic activity for each of 31 different TCGA tumor types and normal tissues. Normal tissue samples are derived both from TCGA and GTEx projects. Boxes in box plot represent interquartile ranges and vertical lines represent 5th–95th percentile ranges, with a notch for the median. p-values are adjusted and calculated by Wilcoxon rank-sum test (comparison to relevant normal). Asterisks (*) denote events significant at 10% FDR. (B) Granzyme A (GZMA) vs perforin 1 (PRF1) expression across TCGA tumor biopsies. Points are colored according to cancer type using the same color coding employed in Figure 1A. Across all cancers, a Spearman rank correlation (r) of 0.87 was observed. (C) Low levels GZMA and PRF1 protein expression detected in tissue microarrays of 20 different tumor types. All representative immunohistochemistry images of each tumor type derived from the Human Protein Atlas.

Figure 2

(A) Metastatic cancers exhibit higher cytotoxic T cell levels. Significantly, increased levels of immune cytolytic activity (CYT) were scored in metastatic skin melanomas, in the TCGA-SKCM dataset. The cytolytic index was also higher in metastatic breast (BRCA) and thyroid cancers (THCA), but did not differ significantly between metastatic and primary tumors. Bars denote mean ± SEM. (B) Pearson’s correlations between CYT and the percentage of tumor-infiltrating lymphocytes and neutrophils, as well as with the percentage of necrosis in primary and metastatic SKCM, BRCA and THCA.

Figure 3

A trend for higher expression (TPM) of a group of genes being expressed in cytotoxic T cell/natural killer (CTL/NK) and non-CTL/NK cells and correlating with cytolytic activity (CYT) in metastatic breast cancers compared to primary tumors. Bars denote mean ± SEM.

Figure 4

Significantly higher expression (TPM) of a group of genes being expressed in cytotoxic T cell/natural killer (CTL/NK) and non-CTL/NK cells and correlating with cytolytic activity (CYT) in metastatic skin melanomas compared to primary tumors. Bars denote mean ± SEM.

Figure 5

A trend for higher expression (TPM) of a group of genes being expressed in cytotoxic T cell/natural killer (CTL/NK) and non-CTL/NK cells and correlating with cytolytic activity (CYT) in metastatic thyroid cancers compared to primary tumors. Bars denote mean ± SEM.

Figure 6

(A) In datasets TCGA-ACC, TCGA-SKCM, and TCGA-BLCA, both individual and simultaneous high levels of perforin 1 (PRF1) and granzyme A (GZMA) were significantly associated with an improved prognosis. On the reverse, simultaneous low expression of both cytolytic genes led to a significant shift toward negative effect vs all other patients. (B) On the contrary, in datasets TCGA-LGG, GSE25066, and TCGA-THYM, accordingly, both individual and simultaneous high levels of GZMA and PRF1 were significantly associated with a worse prognosis, whereas the simultaneous low levels of both genes led to a significant shift toward positive effect vs all other patients. Abbreviations: “ALL.high,” PRF1 high expression and GZMA high expression vs all others; “ALL.low,” PRF1 low expression and GZMA low expression vs all others; “GZMA.ALL,” GZMA high expression vs GZMA low expression; “PRF1.ALL,” PRF1 high expression vs PRF1 low expression.

Figure 7

Overall percentages of lymphocyte and neutrophil infiltration and necrosis (upper part); overall percentage of tumor, normal, and stromal cells (middle part); overall patient survival with respect to the percentage of tumor-infiltrating lymphocytes (TILs) (>5%, high TILs, <5%, low TILs); and representative hematoxylin and eosin slides of ER+, PR+, Her2/neu+ (TPBC) and triple negative breast cancer (lower part).

Figure 8

Overall percentages of lymphocyte and neutrophil infiltration and necrosis (upper part); overall percentage of tumor, normal, and stromal cells (middle part); overall patient survival with respect to tumor-infiltrating lymphocytes and representative hematoxylin and eosin slides of primary and metastatic skin melanomas (lower part).

Figure 9

Overall percentages of lymphocyte and neutrophil infiltration and necrosis (upper part); overall percentage of tumor, normal, and stromal cells (middle part); overall patient survival in respect to tumor-infiltrating lymphocytes and representative hematoxylin and eosin slides of primary and metastatic thyroid carcinomas (lower part).