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. 2024 Oct 15;30(20):4755-4767.
doi: 10.1158/1078-0432.CCR-24-0729.

KIR2DL2/DL3+NKs and Helios+Tregs in Peripheral Blood Predict Nivolumab Response in Patients with Metastatic Renal Cell Cancer

Sara Santagata #  1 , Anna Maria Trotta #  1 , Crescenzo D'Alterio  1 , Maria Napolitano  1 , Giuseppina Rea  1 , Marilena Di Napoli  2 , Luigi Portella  1 , Caterina Ieranò  1 , Giuseppe Guardascione  1 , Elisabetta Coppola  2 , Christophe Caux  3   4 , Bertrand Dubois  3   4 , Helen J Boyle  5 , Joan Carles  6 , Sabrina Rossetti  2 , Rosa Azzaro  7 , Florinda Feroce  8 , Sisto Perdonà  9 , Mario Fordellone  10 , Anna Maria Bello  1 , Daniela Califano  1 , Paolo Chiodini  10 , Sandro Pignata  2 , Stefania Scala  1
Affiliations

KIR2DL2/DL3+NKs and Helios+Tregs in Peripheral Blood Predict Nivolumab Response in Patients with Metastatic Renal Cell Cancer

Sara Santagata et al. Clin Cancer Res. .

Abstract

Purpose: To identify predictive factors of nivolumab sensitivity, peripheral blood NKs and regulatory T-cell (Treg) were evaluated in patients with metastatic renal cell carcinoma (mRCC) enrolled in the REVOLUTION trial.

Experimental design: Fifty-seven mRCCs being treated with nivolumab, as at least second-line of therapy, and 62 healthy donors were longitudinally evaluated (0-1-3-6-12 months) for peripheral NKs and Tregs, phenotype, and function. Multivariable logistic regression was conducted to identify the independent predictors. The 0.632+ internal cross-validation was used to avoid overfitting. The best cutoff value based on a 3-month clinical response was applied to progression-free survival (PFS) and overall survival (OS). Kaplan-Meier curves for PFS and OS were produced.

Results: At pretreatment, mRCCs displayed high frequency of NKp46+NKs, NKp30+NKs, KIR2DL1+NKs, KIR2DL2/DL3+NKs, and PD1+NKs with reduced NK degranulation as well as high frequency of Tregs, PD1+Tregs, Helios+Tregs, and ENTPD1+Tregs. Responder patients, identified as a clinical response after 3 months of treatment, presented at pretreatment significantly low CD3+, high KIR2DL2/DL3+NKs, high PD1+Tregs, and high Helios+Tregs. Upon multivariate analysis, only KIR2DL2/DL3NKs and Helios+Tregs held as independent predictors of nivolumab responsiveness. The KIR2DL2/DL3+NKs >35.3% identified patients with longer OS, whereas the Helios+Tregs >34.3% displayed significantly longer PFS. After 1-month of nivolumab, responder patients showed low CD3+, high NKs, KIR2DL2/DL3+NKs, and ICOS+Tregs. Among these subpopulations, CD3+ and KIR2DL2/DL3+NKs held as independent predictors of nivolumab efficacy. Low CD3+ (≤71%) was significantly associated with longer PFS, whereas high KIR2DL2/DL3+NKs (>23.3%) were associated with both PFS and OS.

Conclusions: Pretreatment evaluation of Helios+Tregs/KIR2DL2/DL3+NKs and 1-month posttreatment CD3+/ KIR2DL2/DL3+NKs will predict nivolumab response in mRCCs.

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Conflict of interest statement

H.J. Boyle reports non-financial support from BMS, Pfizer, Ipsen, and Merck outside the submitted work. J. Carles reports personal fees from Astellas Pharma, AstraZeneca, Bayer, Exelixis, Johnson & Johnson, Novartis (AAA), Pfizer, Sanofi, BMS, Ipsen, and Roche and grants from AB Science, Aragon Pharmaceuticals, Arog Pharmaceuticals, Astellas Pharma, AstraZeneca AB, Aveo Pharmaceuticals, Bayer AG, Blueprint Medicines Corporation, BN Immunotherapeutics, Boehringer Ingelheim España SA, Bristol-Myers Squibb International Corporation (BMS), Clovis Oncology, Cougar Biotechnology, Deciphera Pharmaceuticals, Exelixis, F. Hoffmann-La Roche, Genentech, GlaxoSmithKline SA, Incyte Corporation, Janssen-Cilag International NV, Laboratoires Leurquin Mediolanum SAS, Lilly SA, Medimmune, Millennium Pharmaceuticals, Nanobiotix SA, Novartis Farmacéutica SA, Pfizer SLU, Puma Biotechnology, Sanofi-Aventis SA, SFJ Pharma LTD II, and Teva Pharma SLU outside the submitted work. S. Pignata reports grants and personal fees from MSD, GSK, Roche, and AstraZeneca and personal fees from Novartis outside the submitted work. No conflicts of interest were disclosed by the other authors.

Figures

Figure 1.
Figure 1.
Study design and Kaplan–Meier curves in patients with mRCC. A, Blood samples (24 mL) were collected before nivolumab (baseline, T0) and after 1 (T1), 3 (T3), 6 (T6), and 12 (T12) months of treatment. Clinical assessment every 3 months. B, Study enrollment flow chart. C, Kaplan–Meier curve for PFS and overall survival (OS) in 57 nivolumab-treated patients with mRCC.
Figure 2.
Figure 2.
Pretreatment NKs and Tregs in patients with mRCC-REV. A, Upper: % NKs and % PD1+NKs in HD and REV groups. % NKs: HD (n = 39) vs. REV (n = 54), P = NS; % PD1+NKs: HD (n = 37) vs. REV (n = 51), P < 0.001. Lower: Representative comparison between HD#1457 and REV#4, HD#2 and REV#43. B, Upper: % CD107a+NKs in HD (n = 17) and REV (n = 56) groups, P < 0.05. Lower: Representative comparison between HD#5 and REV#34. C, Upper: % Tregs in HD (n = 34) and REV (n = 55) groups, P < 0.01. % PD1+Tregs: HD (n = 31) vs. REV (n = 51), P < 0.01. Lower: representative comparison between HD#2 and REV#35, HD#1362 and REV#26. D, Upper: % CFSE-Teff proliferation/Tregs dependent in HD (n = 15) vs. REV (n = 55) groups, P < 0.001. Lower: Representative comparison between HD#3 and REV#54. Box plot showing the data distribution across groups with minimum, median, and maximum values. Two-tailed test comparison of differences between two independent groups (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
Figure 3.
Figure 3.
Pretreatment low CD3+, high KIR2DL2/DL3+NKs, PD1+Tregs and Helios+Tregs predict nivolumab response. A, Frequencies of CD3+ (as a percentage of lymphocytes) and KIR2DL2/DL3+NKs, PD1+Tregs, and Helios+Tregs in R vs. NR patients. CD3+: (33 R vs. 31 NR; P < 0.05); KIR2DL2/DL3+NKs: (28 R vs. 21 NR; P < 0.01); PD1+Tregs: (28 vs. 21 NR; P < 0.05); Helios+Tregs: (24 R vs. 17 NR; P < 0.01). Box plot showing the data distribution across groups with minimum, median, and maximum values. Two-tailed test comparison of differences between two independent groups (*, P < 0.05; **, P < 0.01; ***, P < 0.001). B, Kaplan–Meier survival curves for PFS and OS of patients with mRCC-REV, stratified according to optimal cutoff identified by ROC analyses; C, Kaplan–Meier plots for PFS according to combination of Helios+Tregs-KIR2DL2/DL3+NKs values. Low–Low (black line); Low–High (dotted red line); High–Low (red line); High–High (blue line). P-values by log-rank test.
Figure 4.
Figure 4.
One-month nivolumab- CD3+ and KIR2DL2/DL3+NKs predict response. A, Frequencies of CD3+, CD4+, NKs and KIR2DL2/DL3+NKs, and ICOS+Tregs in R vs. NR patients. CD3+ T cells: 33 R vs. 22 NR, P < 0.05; CD4+ T cells: 33 R vs. 22 NR, P < 0.05; NKs: 31 R vs. 21 NR, P < 0.05; KIR2DL2/DL3+NKs: 30 R vs. 21 NR, P < 0.05; ICOS+Tregs: 23 R vs. 19 NR, P < 0.05. Two-tailed test comparison of differences between two independent groups (P < 0.05). Box plot showing the data distribution across groups with minimum, median, and maximum values. Two-tailed test comparison of differences between two independent groups (*, P < 0.05; **, P < 0.01; ***, P < 0.001). B, Kaplan–Meier survival curves for PFS and OS of patients with mRCC-REV stratified according to optimal cutoff identified by ROC analyses. C, Kaplan–Meier plots showing PFS and OS of patients stratified according to a combination of CD3+-KIR2DL2/DL3+NKs. High–Low (black line); High–High (red line); Low–High (blue line); Low–Low (light blue line). P-values by long-rank test.
Figure 5.
Figure 5.
Schematic representative. A, Nivolumab-treated patients with mRCC are evaluated for peripheral NKs and Tregs, phenotype, and function. Clinical evaluation at 3 months identifies patients R and NR. B, Pretreatment (T0) evaluation of NKs and Tregs identified R patients (high KIR2DL2/DL3+NKs and high Helios+Tregs) with improved prognosis. C, One-month post–nivolumab treatment evaluation of NKs and Tregs identified R patients (low CD3+ and high KIR2DL2/DL3+NKs) with improved prognosis.
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