A study at MD Anderson at Cooper showed that out of 305 consecutive NGS assays, only 6 patients started off-label therapies (2%) based on the assay result, and they had a poor prognosis . However, in our study the off-label use of ICIs, when targeting PD-L1 expression or TP53 mutations, consistently met our primary endpoint of stable disease at 16 weeks or later. This finding is consistent with previous studies that demonstrated that PD-L1 and TP53 mutations predict response to ICIs.
A phase 1, non-randomized clinical trial involving 475 patients with 20 types of cancers demonstrated that patients with advanced solid tumors expressing PD-L1 had a higher response rate to pembrolizumab independently of TMB . In addition, a phase 3 randomized, open-label clinical trial with 305 patients with locally advanced or metastatic non-small lung cancer showed that pembrolizumab monotherapy was superior to chemotherapy in adult patients with a PD-L1 TPS of 50% or greater , and another clinical trial with 1274 participants concluded superiority with a PD-L1 TPS of 1% or more . These and other studies led to the FDA approval of pembrolizumab for NSCLC, HNSCC, gastric cancer, esophageal cancer, cervical cancer, and triple-negative breast cancer with different PD-L1 expression thresholds. A study with 72 patients with advanced NSCLC patients treated with programmed death-1 blockers showed higher overall survival in the TP53 mutated group than in the non-mutated group . Other studies with TP53 mutated tumors showed mixed results to immune checkpoint inhibitors and likely different responses of the various TP53 mutations [7, 8, 9].
To characterize off-label use of pembrolizumab we excluded tumors with MSI-H/MSS, MMRd, or TMB ≥10 mut/mb. The most compelling data for its use in these cases was established in KEYNOTE-158. This phase 2 clinical trial enrolled 1595 patients  with 27 different tumor types with a median follow-up of 13.4 months. Patients received pembrolizumab 200 mg IV every three weeks. 233 patients were evaluable for MSI-H/MMRd. The objective response rate (ORR) was 34.3% and 86.9% had a response duration of 12 months or longer . 805 patients were evaluable for TMB, 105 had a TMB ≥10 mut/mb. The ORR was 29% for TMB high patients versus 6% for TMB low .
To our knowledge, no studies have simultaneously analyzed PD-L1 expression, TP53 mutation, MSI, TMB, and MMR status across various tumors with the use of off-label ICIs versus non-ICIs. We also addressed tumors not commonly present on these analyses: papillary, follicular, anaplastic thyroid carcinomas, extramammary Paget's disease of the scrotum, and squamous cell carcinoma of an unknown primary site. Despite the small sample size, the difference between groups was significant suggesting that ICIs may be beneficial in patients with PD-L1 expression or TP53 mutation in the setting of MSS or MSI-H not detected, TMB <10 mut/mb, and MMR proficient tumors not currently FDA approved. This suggests the possibility that off-label use of certain cancer drugs based on NGS may be beneficial for patients without other options of treatment.
This study has limitations. Our sample size was small since we were not conducting a clinical trial but analyzing cases retrospectively of real-world off-label drug use. Mutations that are not currently detected by NGS or not completely understood may have played a role in response to ICIs in the different types of cancer analyzed. For example, a recent study at the MD Anderson Cancer Center with data from over 10,000 tumors included in the Cancer Genome Atlas showed that TMB does not predict response to ICIs equally in different types of cancers . This means that current evidence is insufficient to validate the threshold of 10 mut/Mb and the optimal limit might be higher or lower for different tumor types. Besides, the threshold for PD-L1 expression likely varies among tumors and different TP53 mutations determine a different response to ICIs. Further studies are needed to validate the use of off-label drugs based on NGS, ICIs in tumors with PD-L1 expression or TP53 mutations, and their interrelationship with other predictors of tumor response.