To the editor

In the past decades, neoadjuvant therapy has provided extra treatment opportunities for lung cancer patients and prolonged their survival [1]. For example, in CheckMate-816 [2] trail assessing efficacy of neoadjuvant nivolumab plus chemotherapy on surgically resectable early-stage NSCLC, the combination led to a MPR rate of 36.9% and pathological complete response (pCR) rate of 24%, significantly outperforms the chemotherapy alone arm. However, very few studies reported effective surrogates indicating the outcomes of neoadjuvant immunotherapy in NSCLC. Herein by performing WES on 14 patients receiving immuno-neoadjuvant therapy, we analyzed samples’ genetic changes and their association with patients’ response, aiming to identify indicators for lung cancer immuno-neoadjuvant therapy.

The clinicopathologic characteristics of the patients enrolled are shown in (Additional file 1: Table S1) with variated ages and stages (Additional file 2: Result S1). All patients received immuno-neoadjuvant therapy but with varied regimen combinations (Additional file 3: Fig. S1A-B). Defined as the time interval between first treatment and surgery, neoadjuvant treatment duration did not exhibit significant difference between different responders (Additional file 3: Fig. S1C). As for mutational analyses, 4243 SNVs and 1290 InDels were identified after filtrations and the mutation signature analysis uncovered the enrichment of DDR-related InDel signatures in MPR group (Additional file 2: Result S2). Besides, 293 mutations in 209 cancer driver genes were detected, with TP53 (92.9%), MUC16 (42.9%), CSMD3 (35.7%) and MUC4 (35.7%) most frequently mutated (Fig. 1a). No EGFR/ALK/LKB1 mutations were found in patients. Of particular interest, mutations of tumor suppressor genes from DDR and HR pathways were enriched in MPR patients (Fig. 1b, Additional file 2: Result S2), insinuating the occurrence of HRD events in better-responded group.

Fig. 1
figure 1

Mutational analysis results between MPR and non-MPR groups. a Landscape of frequent SNV and InDel on cancer driver genes. b Pathway enrichment analysis on mutated tumor suppressor genes. c Three HRD metrics including TAI, LST and HRD-LOH calculated on non-aneuploid samples. d HRDscore calculated on non-aneuploid samples. e Correlation between HRDscore and percentage of viable tumor cells in all non-aneuploid samples

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Consistent with the known provocative effect of altered HR pathway (HR) genes on genome instability, the chromosome and arm-level copy number variation (CNV) burden [3] escalated in MPR group and showed negative correlation with percentage of viable tumor cells after treatment with no histological bias (Additional file 2: Result S3, Additional file 3: Fig. S2A-F). Through SCNA signature quantification in non-aneuploid samples (Additional file 3: Fig. S2K), MPR patients were found enriched for signatures correlated with HRD event, emphasizing the association between HR gene alternations and exacerbated SCNA in MPR subpopulation (Additional file 2: Result S3). Possible association between therapeutic benefits and subclonal SCNAs was also observed (Additional file 2: Result S3). The HRD events in MPR patients were further confirmed by the significantly different quantifications of three HRD-related events, either separately (Fig. 1c) or by the summed HRDscore (Fig. 1d) (Additional file 2: Result S3) and such event enrichment persisted in SCC subtype (Additional file 2: Result S3).

We next inspected TMB and ITH considering their recognized impacts on immunotherapy efficacy [4]. Non-aneuploid and high purity MPR samples carried higher TMB (Fig. 2a), which unsurprisingly demonstrated a negative correlation with residual tumor cells (Fig. 2b). When considering the clonality of mutations, disparity between groups exacerbated (Fig. 2c) and clonal TMB decreased with worse therapeutic response (Fig. 2d). Similar conclusions could be drawn on SCC subtype (Additional file 3: Fig. S4A-D). Intriguingly, apart from lower ITH, clonal somatic mutations in HR pathway were enriched in MPR group regardless of subtypes (Fig. 2e), while such trend was not observed in germline HR gene mutations (Additional file 3: Fig. S4E). We also observed the elevated amount of subclonal SCNA in MPR (Fig. 2F, Additional file 2: Result S4) and the subclonality decreased with residual tumor cells (Fig. 2G, Additional file 2: Result S4). Apart from mutational silence, deletion of 13 core HR genes was concentrated in MPR patients (Fig. 2h) and negatively correlated with residual tumor cells (Fig. 2i), indicating activities of HR genes could be synergistically altered at mutation and copy number level, causing SCNA-level ITH fluctuations, and consequently influence the immuno-neoadjuvant clinical outcomes.

Fig. 2
figure 2

Clonal HR pathway gene deactivation impacts neoadjuvant immunotherapy consequences in NSCLC patients. a TMB value distribution between MPR/non-MPR groups. b Correlation between TMB and percentage of viable tumor cells. c Clonal TMB value in two groups. d Correlation between clonal TMB and percentage of viable tumor cells. e Existence of HR pathway genes’ clonal SNV in two groups. The size of the circle was proportional to the mutation number. P values were calculated by Fisher’s exact test on all and SCC samples. f Subclonal SCNA fragment number in two groups. g Correlation between subclonal SCNA fragment number and percentage of viable tumor cells. h Deletion status of 13 HR core pathway genes in MPR/non-MPR groups. P values were calculated by Fisher’s exact test or one-side Wilcoxon rank-sum test. Adenocarcinoma samples were marked with pink triangles. i Correlation between focal deletion number of the 13 genes and percentage of viable tumor cells. j All and clonal neoantigen number generated by two groups. Adenocarcinoma samples were marked with pink triangles. k Clonal TNB value distribution in two groups. l Correlation between clonal TNB and percentage of viable tumor cells

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Additionally, MPR group generated more neoantigens with higher clonal proportion (Fig. 2j, Additional file 2: Result S5). Clonal TNB measuring neoantigen-level ITH was also significantly higher in MPR and inversely correlated with residual tumor cells (Fig. 2k-l, Additional file 2: Result S5). Since neoantigen load could also be affected by HLA class I genes’ activity, more HLA LOH events were found in non-MPR group and the neoantigens on kept HLA alleles anti-correlated with residual tumor cells (Additional file 2: Result S5). Astoundingly, clonal neoantigens on kept HLA alleles were predominantly resided in the amplified SCNAs for MPR patients (Additional file 2: Result S5), prompting the hypothesis that HR gene mutations could instigate SCNA ITH and HLA LOH and eventually orchestrate to compensate the amount of clonal neoantigen, enhancing the response to immuno-neoadjuvant therapy (Additional file 2: Result S5).

Multi-cohort validation was further conducted to confirm the ability of HR gene alternations in therapeutic outcome prediction. In result, HR pathway mutations occurred more frequently in better-responded immunotherapy patients regardless of the treatment regimen and clinicopathologic characteristics. We also observed higher TMB and longer survival brought by HR mutations in immunotherapy patients as well as the substantial amount of HR pathway alternations in multiracial treatment-free samples, which all countenanced the potential of HRD testing as a novel NSCLC immunotherapy biomarker (Additional file 2: Result S6).

In summary, our study associated the deactivation of HR genes and the resulting HRD event with the improved outcomes of immuno-neoadjuvant treatment in NSCLC and validated our discoveries by public cohorts, offering unprecedented guide prior to the armamentarium of immuno-treatments for NSCLC patients.