Next-Generation Sequencing-Based Assessment of JAK2, PD-L1, and PD-L2 Copy Number Alterations at 9p24.1 in Breast Cancer: Potential Implications for Clinical Management

Abstract

Genomic amplification at 9p24.1, including the loci for JAK2, PD-L1, and PD-L2, has recently been described as a mechanism of resistance in postchemotherapy, triple-negative breast cancer. This genomic signature holds significant promise as a prognostic biomarker and has implications for targeted therapy with JAK2 inhibitors, as well as with immunotherapy. To guide future screening strategies, the frequency of these alterations was determined. A total of 5399 cases were included in the study. This encompassed 2890 institutional cases tested by the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets assay and 2509 cases from The Cancer Genome Atlas (TCGA). The combined incidence of 9p24.1 amplifications in both the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets and TCGA cohorts was 1.0% (56/5399 cases) and showed a >10-fold higher incidence in triple-negative breast cancer (triple-negative: 5.1%; non-triple-negative: 0.5%). Tumor mutation burden and stromal tumor infiltrating lymphocytes, parameters used to assess response to immunotherapy, were not significantly higher for these cases. The significance of genomic losses at 9p24.1 is unclear, and further studies are needed. Herein, we studied the spectrum of copy number alterations in breast cancer cases within our institutional clinical sequencing cohort and those profiled by TCGA to determine the frequency of genomic alterations that may predict response or resistance to JAK2 inhibitors and/or immunotherapy.

Figure 1

Immunohistochemistry. Representative hematoxylin and eosin–stained images (A and B) of a triple-negative breast cancer along with corresponding estrogen receptor (C), progesterone receptor (D), human epidermal growth factor receptor 2 (E), and programmed cell death 1 ligand 1 (PD-L1) status (F). PD-L1 shows a diffuse membranous pattern of expression secondary to gene amplification. Original magnification: ×40 (A); ×200 (B–F).

Figure 2

Incidence of 9p24.1 copy number alterations, Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) and The Cancer Genome Atlas (TCGA). The incidences of 9p24.1 amplifications and deletions for breast cancer cases profiled by the MSK-IMPACT and TCGA cohorts combined and corresponding hormone receptor status., CNA, copy number alteration; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; PR, progesterone receptor.

Figure 3

Prognostic factors affecting response to immunotherapy in breast cancer cases with 9p24.1 copy number alterations. A: Tumor mutation burden (x axis) for breast cancers with 9p24.1 copy number alterations. B: No statistically significant trends are observed when cases with 9p24.1 amplifications/deletions are subclassified based on triple-negative status. The dotted vertical line represents the mean tumor mutation burden (TMB) for all breast cancer cases profiled by Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) [3.5 mutations per megabase (mt/MB)]. C: The percentage of cases with 9p24.1 amplifications/deletions identified by MSK-IMPACT in primary or metastatic tumors, before or after chemotherapy. D: The percentage of tumor infiltrating lymphocytes morphologically involving the stromal area (y axis) for primary tumors with 9p24.1 amplifications/deletions. These cases were further subclassified based on whether they received neoadjuvant chemotherapy. Data are expressed as means ± SEM. n = 2890 cases (B); n = 7/10 (D, TNBC cases: n = 2/3 9p24.1Amp-NAC, n = 4/5 9p24.1Amp-No NAC, n = 1/2 9p24.1Del-No NAC). P = 0.08 (D). Amp, amplification; Del, deletion; NAC, neoadjuvant chemotherapy; TNBC, triple-negative breast cancer.

Figure 4

Copy number assessment before and after immunotherapy in a breast cancer patient with 9p24.1 amplification. Copy number plot before (A) and after therapy with pembrolizumab (B) has been shown for the same patient. 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. Black rectangles show probes targeting the 9p24.1 locus to demonstrate amplification that is present only in A and not in B. C: The variant allele frequencies in the specimens before (y axis) and after (x axis) therapy with pembrolizumab. The shared alterations including those involving PIK3CA, TP53, ATM, and NOTCH4 are depicted in red.