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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 (log 2 ratio&gt;0.3) and 19 were classified as non-gain (log 2 ratio≤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 (log 2 ratio=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.

modern pathology

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