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Divergent clinical and immunologic outcomes based on STK11 co-mutation status in resectable KRAS-mutant lung cancers following neoadjuvant immune checkpoint blockade 

Samuel Rosner1,2#, Sydney Connor1,3#, Khaled Sanber1,4, Marianna Zahurak1, Tianbei Zhang1,3, Isha Gurumurthy1,3, Zhen Zeng1,3, Brad Presson1,3, Dipika Singh1,3, Roni Rayes5, Lavanya Sivapalan1, Gavin Pereira1, Zhicheng Ji6, Rohit Thummalapalli7, Joshua E. Reuss1,8, Stephen R. Broderick1, David R. Jones7, Julie S. Deutsch3,9, Tricia R. Cottrell10 Jamie Chaft7,11, Jonathan Spicer5, Janis Taube1,3,9 Valsamo Anagnostou1, Julie R Brahmer1, Drew M. Pardoll1,3, Hongkai Ji12, Patrick M. Forde1, Kristen A. Marrone1¥ and Kellie N. Smith1,3¥ 

Clin Cancer Res November 13 2024

Purpose: Co-mutations of the KRAS and STK11 genes in advanced non-small cell lung cancer (NSCLC) are associated with immune checkpoint blockade (ICB) resistance. While neoadjuvant chemoimmunotherapy is now a standard of care treatment for resectable NSCLC, the clinical and immunologic impact of KRAS andSTK11 co-mutations in this setting are unknown. 

目的：晚期非小细胞肺癌（SOC）中Kras和STK 11基因的共突变与免疫检查点阻断（ICB）耐药性相关。虽然新辅助化疗免疫疗法现在是可切除的非小细胞肺癌的标准治疗方案，但Kras和STK 11共突变在这种情况下的临床和免疫学影响尚不清楚。

Experimental design: We evaluated and compared recurrence-free survival of resectable KRAS-mutated NSCLC tumors, with or without co-occuring STK11 mutations, treated with neoadjuvant ICB. Single cell transcriptomics was performed on tumor-infiltrating T cells from 7 KRASmut/STK11wttumors and 6 KRASmut/STK11mut tumors.

设计：我们评估并比较了接受新辅助ICB治疗的可切除Kras突变的非小细胞肺癌肿瘤（伴或不伴合并STK 11突变）的无复发生存期。对来自7个KRASmut/STK 11 wtt肿瘤和6个KRASmut/STK 11 mut肿瘤的肿瘤浸润性T细胞进行单细胞转录组学。

Results: Relative to KRASmut/STK11wttumors, KRASmut/STK11mut exhibited significantly higher recurrence risk. Single-cell transcriptomics showed enhanced oxidative phosphorylation with evidence of decreased PGE-2 signaling and increased IL-2 signaling in CD8+ tumor-infiltrating lymphocytes (TIL) from KRASmut/STK11mut tumors, a finding that was mirrored in KRASwt tumors that relapsed. TIL from KRASmut/STK11mut tumors expressed high levels of molecules associated with tumor residence, including CD39 and ZNF683 (HOBIT).

结果：相对于KRASmut/STK 11 wttomors，KRASmut/STK 11 mut表现出明显更高的复发风险。单细胞转录组学表现出高氯酸磷酸化增强，有证据表明KRASmut/STK 11 mut肿瘤的CD 8+肿瘤浸润性淋巴细胞（TLR）中PGE-2信号传递减少和IL-2信号传递增加，这一发现也反映在KRASmut/STK 11 mut肿瘤中。来自KRASmut/STK 11 mut肿瘤的TLR表达了高水平的与肿瘤驻留相关的分子，包括CD 39和ZNF 683（HObit）。

Conclusions: These divergent T cell transcriptional fates suggest T cell maintenance and residence may be detrimental to anti-tumor immunity in the context of neoadjuvant ICB for resectable NSCLC, regardless of KRAS mutation status. Our work provides a basis for future investigations into the mechanisms underpinning PGE-2 and IL-2 signaling as they relate to T cell immunity to cancer and to divergent clinical outcomes in KRASmut/STK11mut NSCLC treated with neoadjuvant ICB.

结论：这些不同的T细胞转录命运表明，无论Kras突变状态如何，T细胞的维持和驻留可能会对可切除非小细胞肺癌的新辅助ICB背景下的抗肿瘤免疫力有害。我们的工作为未来研究PGE-2和IL-2信号传递的基础机制提供了基础，因为它们与T细胞对癌症的免疫以及接受新辅助ICB治疗的KRASmut/STK 11 mut非小细胞肺癌的不同临床结果相关。

Statement of Significance: 

This report represents an early, in-depth clinical and immunologic analysis of resectable KRAS-mutant NSCLC treated with neoadjuvant ICB. We show a preliminary signal of divergent clinical outcomes based on the STK11 co-mutation status as well as distinctive phenotypic and metabolic profiles of CD8+ TIL that may underlie these clinical outcomes.

目录

1. INTRODUCTION

2. Materials and Methods

    2.1 Patient selection and eligibility 

    2.2 Treatment procedures (Fig. S2A)

    2.3 Clinical endpoints and biomarkers (Fig. S2B)

    2.4 Statistical Analysis of Clinical Data 

    2.5 Sample Processing 

    2.6 Single cell TCRseq/RNAseq 

    2.7 Single cell data processing and quality control 

    2.8 Single cell data integration and clustering 

    2.9 Single-cell subset pseudobulk gene expression analysis 

    2.10 Differential gene expression profiling 

    2.11 Gene Score generation 

    2.12 Data Availability: 

3. Result

    3.1 Clinical features and outcomes of resectable KRAS-mutated lung cancers treated with neoadjuvant ICB (Fig. S1A)(Tables 1 and S1, Fig. S1B)(Fig. 1)(Fig S2)(Table S2)

    3.2 CD8+ TIL from KRASmut/STK11mut tumors are transcriptionally distinct (Fig. 2)(Table S3)(Fig. S3)

    3.3 CD8+ TIL from co-mutated tumors exhibit features of terminal dysfunction (Fig. 3, Table S4)(Fig. S4)

    3.4 PGE-2 signaling pathways are upregulated in CD8+ TIL from KRASmut/STK11wt tumors (Fig. S5-6)

4. CONCLUSION AND FUTURE PERSPECTIVES 

— 图表汇总—

    3.1 Clinical features and outcomes of resectable KRAS-mutated lung cancers treated with neoadjuvant ICB

Figure S1.  (a) Study schema detailing the various treatment cohorts included in this analysis and corresponding study endpoints and (b) consort diagram detailing the study cohort selection.

Tables 1. Summary of demographic data, neoadjuvant treatment and surgical outcomes for all subjects (n=61) and patients with KRAS mutant disease (n=21). 

a Other histologic diagnoses included pleomorphic and adenosquamous carcinomas.

b Clinical staging was per American Joint Committee on Cancer Tumor Node Metastases 7th edition. 

c On the basis of pre-treatment tumor PD-L1 expression (TPS < 1% vs. ≥1%). There were 10 patients where pre-treatment PD-L1 assessment was not available. 

d 52 subjects with available baseline genomic sequencing 

e KRAS non-G12c mutations in this analysis included: KRAS G12V, KRAS Q61H, KRAS G12D, KRAS G12A, KRAS G12F, KRAS G13C 

Tables S1

Fig. S1B

Fig. 1 Pathologic and clinical outcomes of patients with KRAS-mutant NSCLC treated with neoadjuvant ICB. 

(a) Bar graph presenting the major pathologic response (MPR) rate for patients with KRAS-mutant disease, based on co-mutation status with STK11. The MPR rate for the whole KRAS-mutant cohort is also included as a dotted line for reference and comparison. 

(b) Waterfall plot depicting percent pathologic regression of primary tumor for our KRAS mutant cohort who underwent definitive resection, determined by baseline genomic sequencing prior to neoadjuvant-ICB. Therefore two patients with KRASmut/STK11wt disease and one patient with KRASmut/STK11mut disease were not included as they had primary progression precluding definitive resection. 

(c) Swimmer plot summarizing treatment type and clinical outcomes for all patients with KRAS mutant disease divided by presence or absence of co-occurring STK11 mutation. Patients with primary progression of disease are denoted in this figure, as well as KEAP1 mutation status. 

(d) Kaplan Meier curves depicting recurrence-free survival for patients based on KRAS mutation status. 

(e) Kaplan Meier curves depicting recurrence-free survival for KRAS-mutant cohort based on co-mutation status with STK11.

Figure S2. 

(a) Bar graph describing the major pathologic response rate for patients with resected KRASmutand KRASwt disease. See dotted line which provides reference of major pathologic response rate for all resected patients. (b) Bar graph detailing the partial pathologic response rate for patients with resected KRASmut/STK11wtand KRASmut/STK11mutdisease. See dotted line which provides a reference partial pathologic response rate for all patients with resected KRASmutdisease. (c) Bar graph detailing the complete pathologic response rate for all treated patients with KRASwtand KRASmutdisease, including those without definitive resection, (d) overall survival estimates based on KRAS mutation status, (e) recurrence free survival estimates for patients with KRAS G12C mutations versus non-G12C mutations, (f) recurrence free survival estimates for patients with KRAS mutant disease with or without co-mutation of TP53 and (g) recurrence free survival estimates based on STK11 mutation status.

Table S2

    3.2 CD8+ TIL from KRASmut/STK11mut tumors are transcriptionally distinct

Figure 2. Transcriptional profiling of neoadjuvant ICB-treated CD8+ TIL in NSCLC based on KRAS and STK11 co-mutation status.

(a) Refined clustering was performed on 92,525 CD8+ T cells from tumor (n=13), normal adjacent lung (n=7), and the previously published tissues for MD043-011 which includes tumour-draining lymph node and a distant brain metastasis. Fourteen distinct clusters are annotated and marked by color on the UMAP projection.

(b) Expression of memory, TRM, and T cell checkpoint markers, including CXCL13 and CD39.

(c) Relative expression of the top-5 most differentially expressed genes. Five-thousand cells were randomly sampled from each cluster for visualization.

(d) PCA of cell-cluster-level pseudobulk gene expression for individual tumor samples (n = 13), based on co-mutation status. A one-sided permutation test was performed.  

Table S3

Figure S3. 

(a) CD8 clustering across each individual patient included in this study (b) CD8 clustering representing the cell contributing to analyses performed on KRASmut/STK11mut and KRASmut/STK11wttumor resections. The cell-cluster pseudobulk PCA plot labeled based upon individual patient tumor resection (c), treatment regimen(d), and disease recurrence (e). (f) Cell proportion analysis was performed on CD8+ TIL from KRASmut/STK11mut and KRASmut/STK11wttumor resections. 

    3.3 CD8+ TIL from co-mutated tumors exhibit features of terminal dysfunction

Fig. 3 CD8+ TIL from co-mutated lung cancers exhibit features consistent with terminal differentiation and metabolic dysfunction 

(a) Volcano plot showing differential expression of CD8+ TIL between KRASmut/STK11mut tumors (left) and KRASmut/STK11wt tumors (right). Each dot represents one gene. A false discovery rate (FDR) < 0.05 was considered significant. 

(b) A waterfall plot of the top 10 significantly upregulated genes enriched in KRASmut/STK11mut (red) and in KRASmut/STK11wt (blue) CD8+ TIL. 

(c-e) Violin plots for the expression of TIM-3, LAG3, and TOX2 in CD8+TIL between KRASmut/STK11mut (red) and KRASmut/STK11wt (blue). Comparisons were performed at the individual cell level using two-sided Wilcoxon rank-sum test. 

(f-h) Violin plots for the expression of key genes associated with tissue residence, memory and prostaglandin receptor markers, in CD8+TIL between KRASmut/STK11mut (red) and KRASmut/STK11wt (blue). Comparisons were performed at the individual cell level using two-sided Wilcoxon rank-sum test. 

(i) Feature plot for the OXPHOS score on CD8+ TIL from KRASmut/STK11mut (top) and KRASmut/STK11wt (bottom) tumor resections. This score ranges from 0 to 1. 

(j) Violin plot of the OXPHOS score in CD8+ TIL based on co-mutation status; a two-sided Wilcoxon rank-sum test was performed. 

(k) Violin plot of the expression of IL2RG in CD8+TIL between KRASmut/STK11mut (red) and KRASmut/STK11wt (blue). Comparisons were performed at the individual cell level using two-sided Wilcoxon rank-sum test. 

(l) Violin plot of the IL2 Signaling in CD8+ TIL based on co-mutation status; a two-sided Wilcoxon rank-sum test was performed. 

Table S4 详见文章附件

Figure S4. 

(a) Violin plots showing phenotypic scoring for T cell immune checkpoints, T cell memory, and cytotoxicity for all CD8+ TIL based on co-mutation status. These scores were compared between KRASmut/STK11mutand KRASmut/STK11wtCD8+ TIL using two-sided Wilcoxon rank-sum test. 

(b) Comparison of the OXPHOS activity score between tumor (T) and adjacent normal lung (N) was performed on KRASmut/STK11mut andKRASmut/STK11wt. 

(c) Violin plots for the expression of key genes, including key prostaglandin receptor markers, in CD8+TIL between KRASmut/STK11mut(red) and KRASmut/STK11wt(blue). Comparisons were performed at the individual cell level using two-sided Wilcoxon rank-sum test. 

(d) Violin plot for the expression of PTGER4 in CD8+ TIL the 13 tumor resections based on pathological response status. Comparisons were performed at the individual cell level using two-sided Wilcoxon rank-sum test. 

(e) The mean expression of PTGER4 expression for CD8+ TIL for each individual tumor based upon KRASmut/STK11mut andKRASmut/STK11wtstatus. Each color represents an individual patient tumor resection. Comparisons were performed using a two-sample t-test. 

    3.4 PGE-2 signaling pathways are upregulated in CD8+ TIL from KRASmut/STK11wt tumors

Figure S5. 

(a) Violin plots describing individual gene set expression levels, including key prostaglandin receptor markers, in CD8+ TIL from KRASwttumors published previously11, based on recurrence event. KRASwt CD8+ TIL from patients with recurrent disease (Y) are labeled in orange and those without known recurrent disease (N) are labeled in pink. (b) IL-2 signaling score based on recurrence event for previously reported KRASwt/STK11wttumors. Comparisons were performed at the individual cell level using two-sided Wilcoxon rank-sum test.

Figure S6. 

(a) The OXPHOS activity scoring for KRASmut/STK11mut andKRASmut/STK11wttumors. (b) Gene overlays of PDCD1, LAG3, HAVCR2, TIGIT and ENTPD1 split based upon co-mutation status. (c) The OXPHOS activity scoring between KRASmut/STK11mut andKRASmut/STK11wtfor each CD8+ TIL subtype. 

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