Advanced, anaplastic lymphoma kinase (ALK)-positive lung cancer is currently treated with the first-generation ALK inhibitor crizotinib followed by more potent, second-generation ALK inhibitors (e.g., ceritinib, alectinib) upon progression. Second-generation inhibitors are generally effective even in the absence of crizotinib-resistant ALK mutations, likely reflecting incomplete inhibition of ALK by crizotinib in many cases. Herein, we analyzed 103 repeat biopsies from ALK-positive patients progressing on various ALK inhibitors. We find that each ALK inhibitor is associated with a distinct spectrum of ALK resistance mutations and that the frequency of one mutation - ALK G1202R - increases significantly after treatment with second-generation agents. To investigate strategies to overcome resistance to second-generation ALK inhibitors, we examine the activity of the third-generation ALK inhibitor lorlatinib in a series of ceritinib-resistant, patient-derived cell lines, and observe that the presence of ALK resistance mutations is highly predictive for sensitivity to lorlatinib, whereas those cell lines without ALK mutations are resistant.

he Cancer Genome Atlas project identified 2 groups of endometrioid endometrial cancers (ECs) with high mutation frequency: an ultramutated group (7% of all tumors) that harbored mutations in the exonuclease domain of polymerase e (POLE), and a hypermutated group (28% of tumors) with microsatellite instability (MSI), the majority of which harbored MLH1 promoter methylation. 1 The ultramutated POLE group exhibited an extremely high mutation rate (232 × 10 −6 mutations/Mb) with a unique nucleotide change spectrum of increased C→A transversion frequency, whereas the hypermutated MSI group exhibited mutation rates of 18 × 10 −6 mutations/Mb with variable length of DNA microsatellites due to an underlying deficiency in mismatch DNA repair. [1][2][3] Mismatch DNA repair deficiency induces singlebase mismatches that lead to point mutations in coding regions of genes, as well as insertions or deletions that lead to frame-shift mutations.It has been suggested that hypermutated tumors may harbor more tumor-specific neoantigens and increased amounts of tumor-infiltrating lymphocytes (TILs). 3-7 Therefore, we assessed whether POLE and MSI ECs harbor more neoantigens and TILs than the comparatively hypomutated microsatellitestable (MSS) ECs. Methods Prediction of HLA Type and Neoantigen LoadInference of HLA type was performed using the POLY-SOLVER (polymorphic loci resolver) tool. 5 For prediction of neoantigen load, the Sage Bionetworks' Synapse resource (https://www.synapse.org and Lawrence et al 8 ) and the Net-MHCpan tool (version 2.4) 9 were used (eMethods in the Supplement).IMPORTANCE Immune checkpoint inhibitor therapy has shown benefit in various cancers, but their potential in endometrial cancer (EC) is unknown.OBSERVATIONS Prediction of neoantigen load was performed using sequencing data from the Cancer Genome Atlas data set. Evaluation of tumor-infiltrating lymphocytes (TILs) and PD-1 and PD-L1 expression was performed in 63 patients with EC referred to our institution. The predicted median (range) neoantigen load (predicted neoepitopes per sample

Immune checkpoint inhibitors (e.g., anti-PD-1 and anti-PD-L1 antibodies) have demonstrated remarkable efficacy against hypermutated cancers such as melanomas and lung carcinomas. One explanation for this effect is that hypermutated lesions harbor more tumor-specific neoantigens that stimulate recruitment of an increased number of tumor-infiltrating lymphocytes (TILs), which is counterbalanced by overexpression of immune checkpoints such as PD-1 or PD-L1. Given that BRCA1/2-mutated high grade serous ovarian cancers (HGSOCs) exhibit a higher mutational load and a unique mutational signature with an elevated number of larger indels up to 50 bp, we hypothesized that they may also harbor more tumor-specific neoantigens, and, therefore, exhibit increased TILs and PD-1/PD-L1 expression. Here, we report significantly higher predicted neoantigens in BRCA1/2-mutated tumors compared to tumors without alterations in homologous recombination (HR) genes (HR-proficient tumors). Tumors with higher neoantigen load were associated with improved overall survival and higher expression of immune genes associated with tumor cytotoxicity such as genes of the TCR, the IFN-gamma and the TNFR pathways. Furthermore, immunohistochemistry studies demonstrated that BRCA1/2-mutated tumors exhibited significantly increased CD3+ and CD8+ TILs, as well as elevated expression of PD-1 and PD-L1 in tumor-associated immune cells compared to HR-proficient tumors. Survival analysis showed that both BRCA1/2-mutation status and number of TILs were independently associated with outcome. Of note, two distinct groups of HGSOCs, one with very poor prognosis (HR proficient with low number of TILs) and one with very good prognosis (BRCA1/2-mutated tumors with high number of TILs) were defined. These findings support a link between BRCA1/2-mutation status, immunogenicity and survival, and suggesting that BRCA1/2-mutated HGSOCs may be more sensitive to PD-1/PD-L1 inhibitors compared to HR-proficient HGSOCs.