Development and validation of a novel clinical fluorescence in situ hybridization assay to detect JAK2 and PD-L1 amplification: a fluorescence in situ hybridization assay for JAK2 and PD-L1 amplification

Abstract

The amplification of chromosome 9p24.1 encoding PD-L1, PD-L2, and JAK2 has been reported in multiple types of cancer and is associated with poor outcome, upregulation of PD-L1, and activation of the JAK/STAT pathway. We have developed a novel fluorescence in situ hybridization assay which combines 3 probes mapping to 9p24.1 with a commercial chromosome 9 centromere (CEN9) probe for detection of the JAK2/9p24.1 amplification. JAK2 fluorescence in situ hybridization was compared with array-based comparative genomic hybridization in 34 samples of triple negative breast cancer tumor. By array-based comparative genomic hybridization, 15 had 9p24.1 copy-number gain (logratio>0.3) and 19 were classified as non-gain (logratio≤0.3). Copy-number gain was defined as JAK2/CEN9 ratio ≥1.1 or average JAK2 signals≥3.0. Twelve of 15 samples with copy-number gain by array-based comparative genomic hybridization were also detected by fluorescence in situ hybridization. Eighteen of 19 samples classified as copy-number non-gain by array-based comparative genomic hybridization were concordant by array-based comparative genomic hybridization. The sensitivity and specificity of the fluorescence in situ hybridization assay was 80% and 95%, respectively (P=0.02). The sample with the highest level of amplification by array-based comparative genomic hybridization (logratio=3.6) also scored highest by fluorescence in situ hybridization (ratio=8.2). There was a correlation between the expression of JAK2 and amplification status (Mean 633 vs 393, P=0.02), and there was a trend of association with PD-L1 RNA expression (Mean 46 vs 22, P=0.11). No significant association was observed between PD-L1 immunohistochemistry expression and copy-number gain status. In summary, the novel array-based comparative genomic hybridization assay for detection of chromosome 9p24.1 strongly correlates with the detection of copy-number gain by array-based comparative genomic hybridization. In triple negative breast cancer, this biomarker may identify a relevant subset of patients for targeted molecular therapies.

Expression Patterns, Prognostic Value, and Intratumoral Heterogeneity of PD-L1 and PD-1 in Thymoma and Thymic Carcinoma

“…Expression was graded in a semiquantitative scoring system from 0 to 5 (0 indicating no expression, 5 indicating high expression) as described elsewhere. [28][29][30]…”

Next-Generation Sequencing–Based Assessment of JAK2, PD-L1, and PD-L2 Copy Number Alterations at 9p24.1 in Breast Cancer

“…This is especially true in the case of 9p24.1 amplifications where the shortest region of amplification overlap spans approximately 777 kb including loci for JAK2, PD-L1, and PD-L2, because IHC for markers such as PD-L1 have shown inferior sensitivity when compared with global copy number assessment for the identification of such cases. 14,17 FISH-based methodologies may also represent an effective screening strategy for these tumors because hybridization probes spanning >300 kb may include both the loci for JAK2 as well as PD-L1. 17 However, this approach provides less detailed information regarding multiple genes at the same locus and cannot distinguish focal 9p24.1 gains from larger segmental gains, and does not document other clinically relevant molecular alterations.…”

“…15,16 Given the implications of 9p24.1 amplification in clinical management, recent studies have focused on the development of fluorescent in situ hybridization (FISH)-based strategies to identify these cases in day-to-day clinical practice. 17 To date, the overall incidence of these alterations in breast cancer remains undefined because prior studies have interrogated limited clinical cohorts. 7,14 For instance, studies by Barrett et al 14 and Balko et al 7 identified this signature in 10% (7/68) to 29% (12/41) of TNBC cases.…”

Palmitoylation is critically required for cancer intrinsic PD-1 expression and functions

“…Our group is successful at rationally designing peptide with biological activity and potential therapeutic benefit 19 . To impair with PD-1 palmitoylation, we decided to design a peptide 8 that interfere with the main PD-1 palmitoylation enzyme DHCC9 through competitive inhibition. To this end, we start by the identified sequence surrounding the palmitoylation site of PD-1 at Cys192 (Fig.1a) and subsequently optimized its cell-penetrating potential in silico using to CellPPD predictor 20 .…”

“…Palmitoylation is typically the covalent attachment of palmitic acid to cysteine of membrane proteins, which serves as a mechanism to regulate protein localization and function 8 .…”