JAK2/PD-L1/PD-L2 (9p24.1) amplifications in renal cell carcinomas with sarcomatoid transformation: implications for clinical management

Abstract

Amplifications of JAK2, PD-L1, and PD-L2 at 9p24.1 lead to constitutive expression of PD-L1. This, coupled with JAK2-activation dependent upregulation of PD-L1 and adaptive/induced expression leads to higher tumor PD-L1 expression and immune evasion. Renal tumors were therefore evaluated for 9p24.1 amplifications. A combination of next generation sequencing-based copy number analysis, fluorescence in situ hybridization for JAK2/INSL6 and PD-L1/PD-L2 and immunohistochemistry for phospho-STAT3 (downstream target of JAK2), PD-L1, PD-L2, and PD-1 was performed. In this study we interrogated a "Discovery" cohort of 593 renal tumors, a "Validation" cohort of 398 high-grade renal tumors, The Cancer Genome Atlas (879 cases) and other public datasets (846 cases). 9p24.1 amplifications were significantly enriched in renal tumors with sarcomatoid transformation (5.95%, 15/252) when compared to all histologic subtypes in the combined "Discovery", "Validation" and public datasets (16/2636, 0.6%, p < 0.00001). Specifically, 9p24.1 amplifications amongst sarcomatoid tumors in public datasets, the "Discovery" and "Validation" cohorts were 7.7% (6/92), 15.1% (5/33), and 3.1% (4/127), respectively. Herein, we describe 13 cases and amplification status for these was characterized using next generation sequencing (n = 9) and/or fluorescence in situ hybridization (n = 10). Correlation with PD-L1 immunohistochemistry (n = 10) revealed constitutive expression (mean H-score: 222/300, n = 10). Analysis of outcomes based on PD-L1 expression in tumor cells performed on 282 cases ("Validation" cohort) did not reveal a significant prognostic effect and was likely reflective of advanced disease. A high incidence of constitutive PD-L1 expression in tumor cells in the "Validation" cohort (H-Score ≥250/300) was noted amongst 83 rhabdoid (6%) and 127 sarcomatoid renal tumors (7.1%). This suggests additional mechanisms of constitutive expression other than amplification events. Importantly, two patients with 9p24.1-amplified sarcomatoid renal tumors showed significant response to immunotherapy. In summary, a subset of renal tumors with sarcomatoid transformation exhibits constitutive PD-L1 overexpression and these patients should be evaluated for enhanced response to immunotherapy.

Figure 1:. Copy Number Assessment (Case 1).

A schematic representation of genes present at the 9p24.1 locus is shown ((a) not to scale). A copy number plot for a sarcomatoid renal cell carcinoma profiled using MSK-IMPACT has been depicted (b). Relative (Log2) tumor/normal ratios (y-axis) and corresponding chromosomes (x-axis) are displayed, with each blue dot representing an individual probe region. Amplified regions are shown in red and show a 3.5-fold amplification for JAK2/PD-L1 and PD-L2 at 9p24.1. MSK-IMPACT, Memorial Sloan Kettering Cancer Center Integrated Mutation Profiling of Actionable Cancer Targets.

Figure 2:. Histopathology, Fluorescence In Situ Hybridization and Immunohistochemistry (Case 1 & 4).

Representative H&E-stained images of 9p24.1-amplified sarcomatoid renal cell carcinomas ((a) Case 4, 2.7-fold amplification by MSK-IMPACT, × 400 magnification; (d) Case 1, 3.5-fold amplification by MSK-IMPACT, × 200 magnification), corresponding fluorescence in situ hybridization ((b) Red: PD-L1, Green: PD-L2, Aqua: Centromere; (e) Orange: JAK2, Green: INSL6, Red: Centromere) and immunohistochemistry ((c) PD-L1, × 400 magnification; (f) phospho-STAT3, × 200 magnification) have been depicted. MSK-IMPACT, Memorial Sloan Kettering Cancer Center Integrated Mutation Profiling of Actionable Cancer Targets.

Figure 3:. Histopathology and Immunohistochemistry (Case 4).

Representative H&E stained images of a 9p24.1-amplified clear cell renal cell carcinoma with sarcomatoid transformation is depicted ((a) adjacent clear cell and sarcomatoid areas, × 40 magnification; (c) representative area with clear cell morphology, x 200 magnification; (e) representative area with sarcomatoid morphology, x 200 magnification). Corresponding immunostaining for PD-L1 shows absence of expression in areas with a lower grade clear cell component ((b) left, × 40 magnification; (d) x 200 magnification) and constitutive expression in areas with a higher grade sarcomatoid component ((b) right, × 40 magnification; (f) x 200 magnification).

Figure 4:. PD-L1 Expression Status of High-Grade Renal Cell Carcinomas: Prognostic Significance.

H-scores for PD-L1 expression based on immunohistochemistry was calculated semi quantitatively as the product of intensity of staining (graded from 0 to 3) and the percentage of positive cells showing membranous staining (0 to 100%) for 398 renal cell carcinomas. The range of distribution of H-Scores show significantly higher expression for 9p24.1 amplified renal tumors compared to cases without this genomic signature (a). The percentage of indicated sub-categories of renal tumors that have a H-Score ≥50, ≥100, ≥150, ≥200 and ≥250 is shown (b). Univariable associations of categorized (c) and continuous (d) PD-L1 H-scores with cancer-specific survival are depicted. In addition, univariable associations of categorized (e) and continuous (f) PD-L1 H-scores with distant metastasis-free survival are depicted. S RCC: sarcomatoid renal cell carcinoma; Rh RCC: renal cell carcinoma with rhabdoid features; Amp RCC: 9p24.1 amplified renal cell carcinoma.