Patient characteristics and tumor mutational burden
Of 134 SCLCs which underwent successful targeted NGS with TMB assessment, 52 (38.8%) were treated with ICIs (Additional file 1: Figure S1), and 82 (61.2%) did not receive ICIs for the following reasons: 21 never received any systemic therapy due to poor performance status or because their cancer had not recurred after definitive treatment for limited-stage SCLC; 49 did not receive ICIs because they received treatment between March 2012 and May 2018 prior to the FDA approval of immunotherapy for SCLC and were not able to get immunotherapy on clinical trials; 12 had not progressed on their last systemic treatment prior to the data cut-off. In the ICI-treated cohort, 31 (59.6%) received anti-PD-1 monotherapy (24 received nivolumab; 7 received pembrolizumab) and 21 (40.4%) received nivolumab in combination with ipilimumab. Immunotherapy was administered in the setting of a clinical trial in 22 (42.3%) patients, and 30 patients (57.7%) received commercial immunotherapy. The median age of patients was 65 (range: 43–84) and 94.2% were current or former smokers. In the entire cohort of 134 TMB-evaluable SCLC patients, the median TMB was 9.68 mutations/megabase (mut/Mb) (range: 1.21–31.18), and a similar TMB distribution was observed in the subgroup of 52 ICI-treated patients (median: 9.78, range: 1.33–31.18, Additional file 2: Figure S2). Targeted NGS was performed in all cases on tumor specimens obtained at the time of initial pathologic diagnosis. “TMB high” was defined as cases with a TMB above the 50th percentile (TMB > 9.68 mut/Mb), and “TMB low” was defined as cases at or below the 50th percentile (≤ 9.68 mut/Mb). Baseline clinicopathological characteristics were balanced between the TMB high and TMB low groups, as summarized in Table 1. TMB was also analyzed in tertiles: “TMB upper” (> 12.10 mut/Mb), “TMB middle” (between 12.10 and 8.36 mut/Mb, inclusive), and “TMB lower” (< 8.36 mut/Mb).
Association between TMB and efficacy of immunotherapy
In the cohort of 52 TMB-evaluable and ICI-treated SCLC patients, the objective response rate (ORR) was 15.4% (95% CI: 6.9–28.1%), and the disease control rate (DCR) was 38.5% (95% CI: 25.3–53.0%). With a median follow-up of 24.9 months (95% CI: 15.9-NR), the median PFS (mPFS) was 1.7 months (95% CI: 1.3–2.4), and the median OS (mOS) was 5.9 months (95% CI: 2.7–13.2), Additional file 3: Figure S3 A-B, calculated from the start date of immunotherapy.
We next sought to investigate the association between TMB and clinical benefit from ICIs. Overall there was a significant difference in TMB between patients who experienced a partial response, stable disease, and progressive disease (P = 0.02, Fig. 1a). Patients who experienced a partial response (PR) as their best objective response (BOR) to immunotherapy had a higher median TMB compared to those who had progressive disease (PD) as their BOR (14.83 versus 8.47 mut/Mb). When grouped together, patients who achieved either a PR or stable disease (SD) as their BOR had a significantly higher median TMB compared to those who had PD as their BOR (12.74 versus 8.47 mut/Mb, P < 0.01, Additional file 4: Figure S4). Although there was no significant difference in the ORR between patients in the TMB high group (6 of 26, 23.1%) and the TMB low group (2 of 26, 7.7%, P = 0.25) (Fig. 1b), TMB high patients had a significantly higher DCR compared to TMB low patients (57.7% versus 19.2%, P = 0.01).
We next examined the progression-free and overall survival according to TMB. The mPFS was significantly longer in the TMB high group compared to the TMB low group (3.3 versus 1.2 months, HR: 0.37 [95% CI: 0.20–0.69], P < 0.01, Fig. 2a). In addition, mOS was significantly longer in the TMB high group compared to the TMB low group, whether calculated from the start date of immunotherapy (10.4 versus 2.5 months, HR: 0.38 [95% CI: 0.19–0.77], P < 0.01, Fig. 2b) or from the date of initial SCLC pathologic diagnosis (33.9 versus 15.6 months, HR: 0.39 [95% CI 0.19–0.79], P < 0.01, Additional file 5: Figure S5). Importantly, in a univariate model, we found that gender, baseline brain metastases and type of treatment received (anti PD-1 + anti CTLA-4 versus anti PD-1 monotherapy), were not significantly associated with OS. However, both age (< 70 versus ≥ 70 years, HR: 0.44 [95% CI: 0.22–0.87], P = 0.02) and Eastern Cooperative Oncology Group performance status (ECOG-PS) (ECOG 0–1 versus ≥2, HR: 0.44 [95% CI: 0.22–0.88, P = 0.02) were significantly associated with OS. We then ran a multivariate model with TMB, adjusting for age and ECOG PS, to evaluate whether TMB was still significantly associated with OS. After adjusting for age (< 70 versus ≥70 years, HR: 0.59 [0.28–1.28], P = 0.1801), Eastern Cooperative Oncology Group performance status (ECOG-PS) (ECOG 0–1 versus ≥2, HR: 0.66 [0.30–1.46], P = 0.31) we found that a TMB above median retained a significant association with a longer OS in multivariable analysis (HR: 0.47 [95% CI: 0.22–0.97], P = 0.04). In light of the continuous nature of TMB as variable, we also performed a univariate Cox model with TMB as a continuous variable and found that TMB maintains its significant association with both prolonged PFS (HR: 0.91 [95% CI: 0.85–0.96], P < 0.01) and OS (HR: 0.89 [95% CI: 0.83–0.96], P < 0.01).
To further confirm that TMB is a predictive biomarker only for immunotherapy and not for chemotherapy, we next examined the relationship between TMB and clinical outcomes with chemotherapy. Among the 61 patients with ES-SCLC treated with first-line platinum/etoposide who never received subsequent immunotherapy, there was no association between TMB and mPFS (6.2 versus 6.2 months, HR: 0.72 [95%CI: 0.40–1.30], P = 0.28) or mOS (11.7 versus 10.4 months, HR: 0.84 [95% CI: 0.45–1.57], P = 0.58) when calculated from the start date of first-line chemotherapy (Fig. 2 c-d). Similarly, among the 52 ICI-treated patients, there was no significant difference in mPFS to first-line platinum/etoposide between the TMB high and TMB low groups (6.2 versus 5.6 months, HR: 0.59 [95% CI: 0.34–1.04], P = 0.07, Additional file 6: Figure S6). Lastly we performed a Cox model with an interaction between TMB as a continuous measure and whether or not the patient received immunotherapy. We found that the effect of higher TMB on prolonged overall survival was restricted tothose patients who received immunotherapy, but did not impact survival in patients who never received immunotherapy (P = 0.04).
We next investigated clinical outcomes when SCLCs were stratified by increasing TMB tertiles. We found the mPFS (95% CI) increased from 1.3 (0.9–2.7) to 1.5 (1.0–9.6) to 3.8 (1.6-NR) months, in the lower, middle, and upper tertiles, respectively (P = 0.03), and the mOS (95% CI) increased from 2.5 (2.1–6.8) to 8.0 (1.6–14.1) to 10.5 (5.9-NR) months in the lower, middle, and upper tertiles, respectively (P = 0.02). Consistently, the 1-year survival rates increased along with increasing TMB cutoffs. The 1-year PFS rate was 7.1, 11.1 and 37.1% in the lower, middle, and upper tertiles, respectively, and the 1-year OS rate was 7.1, 40.7, 47.2% in the lower, middle, and upper tertiles, respectively (Fig. 3 a-b).