Targeted inhibitors of neurotropic tyrosine kinases are highly effective in selected patients with gene fusions involving NTRK1, NTRK2, or NTRK3. These fusions are consistently detected in rare cancer types (e.g., secretory breast carcinoma and congenital infantile fibrosarcoma), but the occurrence of NTRK fusions in common cancers and their relationship to other therapy biomarkers are largely unexplored. Tissue samples from 11,502 patients were analyzed for 53 gene fusions and sequencing of 592 genes, along with an immunohistochemical evaluation of TrkA/B/C and PD-L1. Thirty-one cases (0.27% of the entire cohort) had NTRK fusions. The most common fusions were ETV6:NTRK3 (n = 10) and TPM3:NTRK1 (n = 6). Gliomas had the highest number of NTRK fusions (14/982, 1.4%), most commonly involving NTRK2 (n = 9). Seventeen non-glioma cases with NTRK fusions included carcinomas of the lungs, thyroid, breast, cervix, colon, nasal cavity, cancer of unknown primary and soft tissue sarcomas. Strong and uniform Trk expression detected with a pan-Trk immunohistochemistry characterized 7/8 NTRK1 fusion cases and 8/9 NTRK2 fusion cases, while NTRK3 fused cases were positive in 6/11 (55%) of cases. 29% of NTRK fusion cases had no other pathogenic genomic alteration. PD-L1 expression was observed in 23% of NTRK fused cases while high tumor DNA microsatellite instability was detected in two cases. We confirm the rarity of NTRK genes fusions outside the brain malignancies. NTRK inhibitors alone or combined with immune checkpoint inhibitors may be a therapeutic option for a substantial proportion of these patients. Strategies for detection of the NTRK fusion-driven cancers may include immunohistochemistry, but gene fusion detection remains the most reliable tool.

Microsatellite instability (MSI) testing identifies patients who may benefit from immune checkpoint inhibitors. We developed an MSI assay that uses data from a commercially available next‐generation sequencing (NGS) panel to determine MSI status. The assay is applicable across cancer types and does not require matched samples from normal tissue. Here, we describe the MSI‐NGS method and explore the relationship of MSI with tumor mutational burden (TMB) and PD‐L1. MSI examined by PCR fragment analysis and NGS was compared for 2189 matched cases. Mismatch repair status by immunohistochemistry was compared to MSI‐NGS for 1986 matched cases. TMB was examined by NGS, and PD‐L1 was determined by immunohistochemistry. Among 2189 matched cases that spanned 26 cancer types, MSI‐NGS, as compared to MSI by PCR fragment analysis, had sensitivity of 95.8% (95% confidence interval [CI] 92.24, 98.08), specificity of 99.4% (95% CI 98.94, 99.69), positive predictive value of 94.5% (95% CI 90.62, 97.14), and negative predictive value of 99.2% (95% CI, 98.75, 99.57). High MSI (MSI‐H) status was identified in 23 of 26 cancer types. Among 11,348 cases examined (including the 2189 matched cases), the overall rates of MSI‐H, TMB‐high, and PD‐L1 positivity were 3.0%, 7.7%, and 25.4%, respectively. Thirty percent of MSI‐H cases were TMB‐low, and only 26% of MSI‐H cases were PD‐L1 positive. The overlap between TMB, MSI, and PD‐L1 differed among cancer types. Only 0.6% of the cases were positive for all three markers. MSI‐H status can be determined by NGS across cancer types. MSI‐H offers distinct data for treatment decisions regarding immune checkpoint inhibitors, in addition to the data available from TMB and PD‐L1.

To clarify the relationships among TML, MMR, and immune checkpoint expression, we profiled the frequency of shared biomarker phenotypes. On the basis of a variety of potential biomarkers of response to immune checkpoints, only small subsets of glioma patients are likely to benefit from monotherapy immune checkpoint inhibition.

Cancer cells expressing PD-1 ligands (PD-L1/PD-L2) inhibit immune-modulatory T-cell activation facilitating disease progression. Preliminary clinical trials exploring interruption of PD-1/PD-L1 signaling showed benefit in several cancer types. We analyzed the distribution of PD-1-positive tumor-infiltrating lymphocytes (TIL) and cancer cells' expression of PD-L1 in a molecularly profiled cohort of 437 malignancies (380 carcinomas, 33 sarcomas, and 24 melanomas). We showed that the presence of PD-1 þ TILs significantly varied among cancer types (from 0% in extraskeletal myxoid chondrosarcomas to 93% in ovarian cancer), and was generally associated with the increased number of mutations in tumor cells (P ¼ 0.029). Cancer cell expression of PD-L1 varied from absent (in Merkel cell carcinomas) to 100% (in chondro-and liposarcomas), but showed the inverse association with the number of detected mutations (P ¼ 0.004). Both PD-1 and PD-L1 expression were significantly higher in triple-negative breast cancers (TNBC) than in non-TNBC (P < 0.001 and 0.017, respectively). Similarly, MSI-H colon cancers had higher PD-1 and PD-L1 expression than the microsatellite stable tumors (P ¼ 0.002 and 0.02, respectively). TP53-mutated breast cancers had significantly higher PD-1 positivity than those harboring other driver mutations (e.g., PIK3CA; P ¼ 0.002). In non-small cell lung cancer, PD-1/PD-L1 coexpression was identified in 8 cases (19%), which lacked any other targetable alterations (e.g., EGFR, ALK, or ROS1). Our study demonstrated the utility of exploring the expression of two potentially targetable immune checkpoint proteins (PD-1/PD-L1) in a substantial proportion of solid tumors, including some aggressive subtypes that lack other targeted treatment modalities. Cancer Epidemiol Biomarkers Prev; 23(12); 2965-70. Ó2014 AACR.