Wedding of Molecular Alterations and Immune Checkpoint Blockade

Genomics as a Matchmaker

Elena Fountzilas, MD, PhD; Razelle Kurzrock, MD; Henry Hiep Vo, PhD; Apostolia-Maria Tsimberidou MD, PhD


J Natl Cancer Inst. 2021;113(12):1634-1647. 

In This Article

FDA-approved Biomarkers

On May 23, 2017, the FDA granted accelerated approval to pembrolizumab for dMMR/MSI diverse solid cancers (Table 1). This was a hallmark approval because, for the first time, a regulatory agency approved an immunotherapy drug based on a genomic marker and, importantly, that approval was tissue agnostic. On June 16, 2020, the FDA gave a second similar (histology-agnostic) approval to pembrolizumab. This time, the approval was for solid tumors harboring TMB of at least 10 mut/mb. It is important to note that the vast majority of tumors with dMMR/MSI have a high TMB, but most tumors with a high TMB are not MSI. Therefore, it is probable that high TMB mediates the responsiveness to checkpoint blockade, perhaps by resulting in a high number of mutanome-derived neo-antigens, hence increasing the chances that one or more of these neo-antigens will be immunogenic and trigger T-cell activation, leading to eradication of the cancer cells bearing the immunogenic neo-antigens.

dMMR and Microsatellite Instability

MMR proteins control the excision of DNA mismatches introduced by DNA polymerase during cell division; these mismatches commonly occur in repetitive DNA sequences known as microsatellites. Impairment of the MMR system leads to MSI, which is characterized by the accumulation of mismatches in repeated sequences. Defects in the MMR system can be assessed using polymerase chain reaction–based assays that test for MSI or immunohistochemical (IHC) analyses of the expression of MMR proteins, including MLH1, MSH2, MSH6, and PMS2. Both tests are approved for selecting patients for treatment. MSI-high (MSI-H) or dMMR tumors can arise because of somatic or epigenetic MMR gene alterations[53,54] or to Lynch syndrome-associated germline MMR gene mutations.[55–57]

The genomic analysis of more than 15 000 cancers (>50 types of malignancy) revealed MSI-H in 2.2% of specimens of which 16% had a germline mutation in an MMR gene, consistent with Lynch syndrome.[58–60] MSI-H has been identified in 28% of patients with endometrial cancer and in 19% of patients with colon cancer.[58,59] It is also reported in diverse tumor types (not classically considered in Lynch syndrome), including gastric (22%), adrenal cortical (5%), and esophageal (3%) carcinomas.[58]

Several trials have shown statistically significant responses to anti-programmed cell death-1 (PD-1) agents in heavily pretreated patients with MSI-H/dMMR in diverse tumor types, leading to FDA approval of MSI/dMMR as a predictive biomarker for immune checkpoint blockade. Notably, in May 2017, pembrolizumab received the first tissue and site-agnosticFDA approval for the treatment of adult and pediatric patients with unresectable or metastatic MSI-H or dMMR solid tumors that had progressed after prior treatment.[7] Five single-arm trials evaluated the use of pembrolizumab in patients with dMMR or MSI-H tumors; the overall response rate (ORR) was 39.6% (complete response [CR] = 7%), with a large subgroup of responses being durable.[6,10,61,62] Additionally, pembrolizumab was approved for the treatment of unresectable or metastatic MSI-H or dMMR colorectal cancer (CRC) that had progressed after treatment with a fluoropyrimidine, oxaliplatin, and irinotecan.[63] In June 2020, pembrolizumab was further approved for the first-line treatment of patients with advanced MSI-H or dMMR CRC, based on the results of a multicenter, open-label, randomized trial (KEYNOTE-177).[64] Patients who received treatment with pembrolizumab had longer median progression-free survival (PFS) compared with patients who received standard chemotherapy (16.5 vs 8.2 months; hazard ratio [HR] = 0.60, 95% confidence interval [CI] = 0.45 to 0.80, P = .0002). The ORR was 43.8% with pembrolizumab vs 33.1% with chemotherapy, whereas median duration of response was not reached with pembrolizumab vs 10.6 months with chemotherapy.[64]

Nivolumab, another anti-PD-1 agent, was also granted FDA approval as a monotherapy and in combination with ipilimumab for the treatment of patients 12 years of age or older with MSI-H or dMMR metastatic CRC after prior treatment with a fluoropyrimidine, oxaliplatin, and irinotecan.[65,66] Both approvals were based on the results of CHECKMATE-142, a nonrandomized phase II trial evaluating nivolumab with or without other anti-cancer drugs. Patients with MSI-H/dMMR CRC after 1 or more lines of treatment received nivolumab until disease progression, death, or unacceptable toxicity.[50] Among the 74 patients studied, the ORR was 31%, and the median depth of response was not reached. In the combination arm, patients received nivolumab every 2 weeks in combination with low-dose ipilimumab (1 mg/kg) every 6 weeks. The ORR was 55%, and the median duration of response was not reached; the 12-month PFS rate was 71%.[11] In an updated analysis, the ORR was 60% (95% CI = 44.3 to 74.3); median PFS and overall survival (OS) were not reached.[12] These data demonstrate that MSI-H/dMMR is a powerful marker for response to pembrolizumab and nivolumab in diverse tumor types.

TMB of at Least 10 Mut/mb

TMB is defined as the number of somatic synonymous and nonsynonymous mutations (base substitutions and indels) per megabase of analyzed DNA. Nonsynonymous molecular alterations lead to transcription of abnormal proteins, which are recognized by the immune system as neoantigens and render the tumor highly immunogenic. In June 2020, the FDA granted accelerated approval to pembrolizumab for the treatment of patients with advanced solid tumors with intermediate to high TMB (ie, ≥10 mut/mb) whose disease had progressed on prior treatment and who had no satisfactory alternative treatment options.[9] Additionally, the FoundationOneCDx assay was also approved by the FDA as a companion diagnostic test for pembrolizumab. This approval was based on the results of KEYNOTE-158, a nonrandomized clinical trial of patients with diverse solid tumor types who received treatment with pembrolizumab until disease progression or unacceptable toxicity.[67] The trial aimed to evaluate predictive biomarkers through a prospectively planned retrospective analysis of 10 cohorts of patients. Among 102 patients with tumors with TMB of at least 10 mut/mb, the ORR (primary endpoint) was 29% (CR = 4%), and about half of the responses were durable at 2 years.[67]

An analysis of 104 814 tumor tissue samples from the Foundation Medicine database showed that TMB of at least 10 mut/mb was identified in about 13% of all solid tumors.[68–70] Other investigators retrospectively reviewed data on 1638 patients with diverse tumor types who had undergone comprehensive genomic profiling and reported that TMB-high (TMB-H) tumor status, which was defined as at least 20 mut/mb, was more commonly noted in patients with melanoma (34%) and NSCLC (24%).[8] In that study, among patients who received immunotherapy, TMB-H was independently associated with a higher ORR (58% vs 20%; P = .0001) and longer median PFS (12.8 vs 3.3 months; P < .0001) and OS (not reached vs 16.3 months; P = .0036). Several additional studies have shown a statistically significant clinical benefit of checkpoint inhibitor treatment in patients with higher TMB.[13,14] In patients with NSCLC and TMB of at least 10 mut/mb, frontline nivolumab plus ipilimumab was associated with longer PFS compared with chemotherapy, irrespective of the PD-L1 expression level.[13] Next-generation sequencing (NGS) of 148 803 samples from the University of California San Diego and Foundation Medicine databases further revealed that 9762 (6.6%) were TMB of at least 20 mut/mb, and 2179 (1.5%) were MSI-H.[69] Among 60 patients who received immunotherapy, the median PFS was longer in patients with low MSI and TMB of at least 20 mut/mb compared with those with low MSI and TMB of less than 20 mut/mb (26.8 vs 4.3 months; P = .0173). Additionally, higher TMB was linearly correlated with better outcomes after immune checkpoint blockade,[8] and the impact of TMB was independent of microsatellite status.[69]

Despite robust data demonstrating the association of higher TMB with response to immunotherapy, several challenges remain. First, harmonization of TMB assays is critical. Some investigators suggest that TMB response thresholds might differ across tumor types.[70] Additionally, neoantigen immunostimulation differs on the basis of the corresponding molecular alteration, with certain mutations likely inducing a strong immune response.[71] Further, not all studies show a survival benefit for higher TMB, even when there are robust and durable responses. Importantly, current TMB evaluation algorithms do not take into consideration neoantigens that might be generated from posttranslational modifications or gene fusions. Furthermore, TMB calculation using circulating tumor DNA analysis is being assessed. In patients with advanced NSCLC treated with pembrolizumab, plasma analysis using a 500-gene panel demonstrated that TMB of at least 16 mut/mb was associated with longer PFS.[72] Other biologic considerations such as major histocompatibility complex (MHC) presentation and T-cell receptor (TCR) repertoire may also be important.

PD-L1 Immunohistochemistry

PD-L1 expression is being evaluated in numerous clinical trials as a biomarker predicting response to immunotherapy agents. High PD-L1 membrane expression is assessed by immunohistochemistry and defined using various scoring methods and cutoff points ranging from more than 1% to more than 50%. PD-L1 expression has been approved by the FDA as a companion diagnostic for the administration of immune checkpoint inhibitors in various indications, including first-line treatment for advanced NSCLC, head and neck squamous cell carcinoma, urothelial carcinoma, and triple-negative breast cancer and second-line therapy and beyond for advanced NSCLC, esophageal squamous cell carcinoma, cervical cancer, and gastric-esophagogastric junction adenocarcinoma. High PD-L1 expression has been associated with clinical benefit in patients with selected tumor types.[73–76] This variability in the predictive value of PD-L1 expression and in the cutoff used to determine PD-L1 expression positivity is attributed to differences in tumor type, disease stage, types of checkpoint inhibition (PD-1 vs PD-L1), and methodology. Methodological differences are associated with technical issues regarding assessment of PD-L1 expression in tumor cells and immune cells; the use of scoring systems combining expression in tumor cells, lymphocytes, and macrophages; and the antibodies used for biomarker evaluation. Examples of monoclonal antibodies used to assess PD-L1 expression include SP142 and SP263 (Ventana Medical Systems, Oro Valley, Arizona, USA) and 22C3 pharmDx (Agilent, Santa Clara, California, USA). Scoring methods include the combined positive score, defined as the number of PD-L1–positive cells (tumor cells, lymphocytes, and macrophages) divided by the total number of cells (tumor cells, lymphocytes, and macrophages) and multiplied by 100, and the tumor proportion score (TPS), defined as the number of PD-L1-positive tumor cells divided by the total number of tumor cells and multiplied by 100. To overcome the variability in PD-L1 assessment, standardization of methodology is warranted.