KIR-HLA Functional Repertoire Influences Trastuzumab Efficiency in Patients With HER2-Positive Breast Cancer

Abstract

Trastuzumab induced a high rate of pathological Complete Response (pCR) in patients affected by locally advanced HER2-positive Breast Cancer (HER2-BC), by exploiting immune-mediated mechanisms as Antibody-Dependent Cell Cytotoxicity (ADCC) involving Natural Killer (NK) cells. Host's immune genetics could influence the response to therapy, through the expression of variants that characterize NK receptors involved in ADCC effectiveness. Killer cell immunoglobin-like receptors (KIRs) modulate NK cell activity through their binding to class-I Human Leukocyte Antigens (HLA). The impact of the KIR/HLA repertoire in HER2-BC is under study. We characterized KIR genotypes of 36 patients with locally advanced HER2-BC treated with neoadjuvant chemotherapy including trastuzumab. We monitored pCR achievement before surgery and Disease-Free Survival (DFS) and Overall Survival (OS) after adjuvant therapy. HLA, and Fc gamma receptor IIIa (FcγR3A) and IIa (FcγR2A) were genotyped through targeted PCR and Sanger sequencing in 35/36 patients. The KIR-HLA combinations were then described as functional haplotypes and divided in two main categories as inhibitory tel A and stimulatory tel B. Trastuzumab-dependent ADCC activity was monitored with an in vitro assay using a HER2-BC model and patients' NK cells.We observed a higher frequency of KIR activators in patients who achieved a pCR compared to partial responders. During the study of functional haplotypes, individuals carrying a tel B haplotype showed greater ADCC efficiency than tel A cases. In subjects with the tel A haplotype the presence of the favorite V allele in FcγR3A receptor improved their low ADCC levels. Regardless of the haplotypes detected, the presence of KIR3DL2/HLA-A03 or A11 was always associated with the FcγR3A V allele, and therefore correlated with greater ADCC efficiency. However, this particular KIR receptor appeared to harm DFS and OS. Indeed, patients with tel B haplotype without KIR3DL2/HLA-A03 or A11 showed a better outcome. Our data, although preliminary, suggested a potential predictive role for KIR haplotype tel B, in identifying patients who achieve a pCR after neoadjuvant treatment with trastuzumab, and supported a negative prognostic impact of KIR3DL2/HLA-A03 or A11 in the adjuvant setting.

Trial registration: ClinicalTrials.gov NCT02307227.

Keywords: ADCC; HLA; KIR; breast cancer; trastuzumab.

Figure 1

The histogram shows the average number of activating KIR genes for each individual as a function of the pathological Complete Response (pCR) (n=16) and pathological Partial Response (pPR) (n=20) to Neoadjuvant Chemotherapy (NC) treatment ( P=0.125). The number of activating KIRs appeared higher in patients who achieved pCR. P-value was calculated using the ANOVA test. Bar height and error bars represent the mean ± standard error mean (SEM) for the data set.

Figure 2

Distribution of KIR genes by frequency in the HER2-positive breast cancer in complete (pCR) and partial (pPR) response to neoadjuvant chemotherapy. The frequency of KIR activators (2DS1, 2DS3, 2DS5, 3DS1) was higher in patients who had a pCR. Framework gene KIR 3DL2, as well as a unique inhibitory KIR 2DL5 gene for which a definite ligand is still unknown, were also more frequent in the pCR group.

Figure 3

(A) Figure shows the percentage of normalized Antibody-Dependent Cell Cytotoxicity (ADCC) mediated by NK cells, carrying the tel A genotype and the tel B genotype, in the presence of trastuzumab. Each histogram represents the extent of normalized lysis of calcein-labeled MDA-MB453 cells in the presence of trastuzumab and 10,000 NK cells as effector cells. A higher ADCC efficacy is mediated by effector cells of patients carrying the tel B KIR genotype compared to those obtained from patients with tel A KIR genotype (P=0.07: Bar height and error bars represent the mean ± standard error of the mean (SEM) for the data set. The P-value was calculated using the ANOVA test. (B) Histograms represent the percentage of normalized ADCC mediated by NK cells of patients carrying the KIR tel A genotype (n=19) according to V and F polymorphic FcγRIII variants. Higher cytolysis was observed in patients carrying the tel A genotype in the presence of FcγRIII V-allele compared to F-allele. In detail, we observed the following percentage of normalized ADCC: 4.6 (95%CI -4.33-13.55) in the presence of the homozygous FcγRIII F variant (F/F); 17.06 (95%CI 8.70-25.43) in cases of heterozygous FcγRIII variants (V/F); and 21.9 (95%CI 8.3-35.6) for the homozygous FcγRIII V variant (V/V) carriers. The P value of Jonckheere-Terpstra trend was significant(p=0.0080). Bar height and error bars represent the mean ± standard error of the mean (SEM) for the data set. (C) Histogram report the percentage of normalized ADCC according to KIR telA versus telB genotype, and according to FcγR3A F>V and FcγR2A R>H polymorphic variants. A gradual increase of the percentage of normalized ADCC was observed in patients carrying the FcγR3A V polymorphism, with a higher affinity for the IgG1-Fc, and the inhibitory tel A genotype. The effect of the FcγR2A R variant, with a reduced affinity for IgG1-Fc, was limited. (Jonckheere-Terpstra trend test, p=0.0018). The percentages of normalized ADCC were respectively: 1.98 for FcγR3A- FcγR2A (F-H);, 5.62 for (F-R), 10.26 for (V-H); and 17.65 for (V-R). Conversly, normalized ADCC measured in patients carrying the tel B genotype showed that neither FcγR3A nor FcγR2A polymorphisms had a significant effect (Jonckheere-Terpstra trend test, p=0.8455). V= FcγR3A V/V or V/F genotypes; F= FcγR3A F/F genotype; H= FcγR2A H/H or H/V-genotypes; R= FcγR2A R/R-genotype. Each data represent three replicates. Bar height and error bars represent the mean ± standard error of the mean (SEM) for the data set.

Figure 4

(A) Comparison of normalized Antibody-Dependent Cell Cytotoxicity (ADCC) mediated by NK cells obtained from patients carrying the functional KIR3DL2+/HLA-A*03 or A*11 combinations and from all of the other patients carrying other KIR/HLA combinations. Each histogram represents the extent of normalized lysis of calcein-labeled MDA-MB453 cells in the presence of trastuzumab and 10,000 NK cells as effector cells. Bar height and error bars represent the mean ± standard error of the mean (SEM) for the data set. A higher ADCC efficacy is mediated by effector cells of patients carrying the KIR3DL2+/ligand compared to effectors obtained from patients characterized by other KIR/HLA combinations (ANOVA test, P = 0.036). (B) Interactive action of KIR tel A or B genotype and KIR/HLA combination on ADCC efficacy showed by two ways Anova. A significant increase in cytolysis was observed by using effector cells obtained from patients carrying the tel A-Func genotype in the presence of KIR3DL2+/HLA-A*03 or A*11 compared to those obtained from patients with other KIR/ligand combinations. [% ADCC 11.94 (95%CI 5.58-18.31); % ADCC 21.10 (95%CI 2.54-39.65), t-test p=0.004]. Bar height and error bars represent the mean ± standard error of the mean (SEM) for the data set. (C) An increased effect on ADCC efficacy was observed in the presence of effector cells obtained from patients carrying both the FcγR3A V-allele (Fc-V) and the KIR3DL2+/HLA-A03 or A*11 compared to those carrying the FcγR3A F-allele (Fc-F) and other KIR/HLA combinations [% ADCC =3.47 vs 19.21, p<0.05]. A significant difference was also observed between other KIR/Ligand combinations according to the FcγR3A allele [% ADCC others+Fc-F = 3.47 others +Fc-V= 11.06, Kruskal-Wallis test P <0.05].

Figure 5

(A) Influence of the KIR tel B genotype on CD16 and CD56 membrane expression at 2, 6, and 12 months after surgery. Mononuclear peripheral blood cells from KIR tel genotyped patients (n=10) were labeled with anti-CD3 and anti-CD16 monoclonal antibody and anti-CD56 antibody and analysed by flow cytometry. An increase in the expression of CD16 on NK cells carrying the tel B genotype was found during time (trend P=0.11) (A) Influence of the potential functional KIR3DL2+/HLA-A*03 or A*11 gene combinations on CD16 and CD56 membrane expression at 2, 6, and 12 months after surgery. Mononuclear peripheral blood cells from KIR tel genotyped patients were labeled toghther with anti-CD3, anti-CD16 , and anti-CD56 antibodies and analysed by flow cytometry. Differences among groups were not statistically significant.

Figure 6

Kaplan-Meier survival analysis showed improved Disease-free survival (DFS) time for tel B genotype carriers compared to tel A carriers (A) and absence of the functional tel KIR3DL2+/HLA-A*03 or A*11 combinations compared to other KIR/HLA combinations (B). (C) Co-presence of Tel B genotype and KIR3DL2+/HLA-A*03 or A*11 genes reduce DFS compared to Tel-B lacking the KIR3DL2+/HLA-A*03 or A*11 combinations (HR: 3.85, 95%CI 0.72-20.5, p = 0.1141).

Figure 7

In Breast Cancer (BC) patients treated with Neoadjuvant Chemotherapy (NC), the haplotype including the functional tel B genotype and the KIR3DL2+/ligand was significantly associated with a poor Overall Survival (OS) compared to tel B haplotype without the 3DL2+/ligand as the reference (P = 0.0328), a trend toward a lower-survival was found using Tel A without the 3DL2+/ligand combination as the reference (HR: 2.66, 95%CI 0.47-14.8, P=0.26).