Human leucocyte antigen class I in hormone receptor-positive, HER2-negative breast cancer: association with response and survival after neoadjuvant chemotherapy

The neoadjuvant GeparTrio pilot [17] (NCT00544765) and main (NCT00544765) trials [14, 15] were prospective, randomized phase II and III trials including 2357 patients with breast cancer (cT2–4 cN0–3 cM0) recruited between 2001 and 2005. Patients received two cycles of docetaxel, adriamycin, and cyclophosphamide (TAC) and response was evaluated by ultrasound. Responders received four more cycles of TAC (pilot study) or were randomly assigned to four or six cycles of TAC (main study). Non-responders were randomized to receive either four cycles of TAC or four cycles of vinorelbine and capecitabine. HR+ was defined as ≥ 10% of tumor cells with estrogen receptor and/or progesterone receptor expression, as defined in the study protocol. HER2 positivity was determined by immunohistochemistry (HER2 score 3+) and in situ hybridization where appropriate (HER2/CEP17 ratio > 2.2). Endocrine treatment for 5 years was planned for patients with HR+ disease but was not part of the protocol. HER2 therapy was not available at that time. We used all samples with available material in the central biobank. Additional file 3: Table S1 lists the pathological and clinical characteristics of the patients. The extend of tumor-infiltrating lymphocytes (TILs) was estimated on H&E-stained whole tissue slides as the area of tumor cells (for intratumoral TILs) or the stromal area (for stromal TILs) that is covered by lymphocytes [3].

Immunohistochemical staining was performed on tissue microarrays using the anti-HLA class I antibody EMR-8-5 (dilution 1:600) recognizing the HLA class I HC HLA-A, HLA-B, and HLA-C (MBL, Woburn, MA, USA). The percentage of cells with membranous staining relative to all cancer cells was assigned to five categories: (0) 0%, (1) 1–9%, (2) 10–50%, (3) 51–80%, and (4) 81–100%. The intensity of staining was assigned into four categories: (0) negative, (1) weak, (2) moderate, (3) strong. The groups for percentage and staining intensity were multiplied resulting in the immunoreactive score (IRS) ranging from 0 to 12. Based on the distribution of staining levels and patient outcome, a cut point (IRS > 2) was selected to optimize separation of curves in Kaplan-Meier analysis. In addition, the percentage of positively stained tumor cells was used continuously to evaluate the effect of HLA immunohistochemistry on clinical endpoints without a defined cut-off.

Gene expression data and clinical information of the MD Anderson Cancer Center cohort [18] (Affymetrix U133A microarrays) was downloaded from the GEO repository (GSE25066). A total of 320 tumors were HR+/HER2− and patients received neoadjuvant chemotherapy and adjuvant endocrine treatment if HR+. HR positivity was defined as any number of stained cells. We applied an additional filter based on gene expression of ESR1 (probeset 205225_at) based on its bimodal distribution (> 10.45). In total, 267 patients met these criteria. Additional file 3: Table S2 lists the patient characteristics. HLA-A, HLA-B, and HLA-C metagenes were calculated as mean expression of the respective probesets (HLA-A: 215313_x_at, 213932_x_at; HLA-B: 211911_x_at, 209140_x_at, 208729_x_at, HLA-C: 208812_x_at, 216526_x_at, 214459_x_at, 211799_x_at). Unsupervised cut-offs were chosen by assigning the same fraction of cases to the groups with high (60%) and low (40%) expression as in the GeparTrio dataset (HLA-A > 14.24, HLA-B > 13.63, and HLA-C > 13.69). Probesets 205225_at (estrogen receptor 1) and 208079_s_at (Aurora kinase A) were used to evaluate their association with HLA-A. Immune cell metagenes were calculated as the mean expression of cell-type-specific genes [16].

Pathologic complete response (pCR) was defined as the absence of invasive cancer in breast and lymph nodes (ypT0/ypTis ypN0). In the MD Anderson cohort, response to chemotherapy was recorded using the residual cancer burden index [19]. Disease-free survival (DFS) was defined as time from study entry to local or distant recurrence or death from any cause, distant recurrence-free survival (DRFS) as the interval between diagnosis and distant recurrence or death from any cause, and overall survival (OS) as the time from study entry to death from any cause. To evaluate associations between clinical and pathological variables with HLA I, χ² tests were used. Kaplan-Meier estimators and log-rank tests were used for survival analyses (R package survival). The independent prognostic value was assessed in multivariate Cox regression analyses and likelihood ratio tests. Statistical computations were performed in R version 3.3.1 (R Development Core Team, Vienna, Austria). Significance was based on P <  0.05 and 95% CI estimates.

A total of 732 cases from the GeparTrio trial were analyzed for HLA class I HC expression. In total, 669 cases (91%) showed a positive staining; 63 were negative (9%, Fig. 1). We defined a cut-off to dichotomize HLA class I expression based on data distribution and patient outcome. This resulted in 480 cases with high expression (66%) and 252 (34%) with low expression across breast cancer subtypes. Additional file 3: Table S1 lists the patient and tumor characteristics of the GeparTrio study cohort that was evaluated for HLA class I HC expression (and Additional file 3: Table S2 the characteristics of the HR+/HER2− cohort).

The frequency of HLA class I HC expression was higher in TNBC compared to other subtypes (81% in TNBC, Fig. 1), in patients with node-positive disease (P = 0.004) and in tumors with a higher histological grade (P <  0.001). High levels of HLA class I HC expression was associated with extensive stromal TILs in the entire cohort (P < 0.001, Fig. 1) and within HR+/HER2− tumors (P < 0.001), where both the extent of intratumoral and stromal TILs were associated with high HLA class I HC expression (Fig. 2). There was no significant association with HER2 status, clinical T stage (T1–2 vs. T3–4), and histological subtype (no special type vs. lobular; data not shown).

High HLA class I HC expression was associated with a higher rate of pathologic complete response (pCR) in patients with HR+/HER2− disease (7% vs. 14%, P = 0.029, Table 1), even after adjustment for clinical and pathological characteristics, but not in other subtypes (Table 1). High HLA class I expression was associated with shorter DFS in HR+/HER2− breast cancer (P = 0.024, Fig. 3, Table 1), but not in other subtypes (Table 1). In patients with HR+/HER2− breast cancer, HLA class I HC expression was prognostic when stratified for nodal status (Fig. 3) and in a multivariate analysis adjusted for clinical and pathological characteristics (Table 1). In an exploratory analysis within HR+/HER2− breast cancer, HLA class I HC was associated with higher risk of relapse in patients with grade 1–2 tumors, patients with residual disease after neoadjuvant chemotherapy, in patients with non-lobular breast cancer and those without extensive intratumoral lymphocytes (Additional file 1: Figure S1). We also used the percentage of cells positively stained for HLA I to evaluate its association with clinical endpoints without a predefined cut-off (Additional file 3: Table S3).

For validation, we used data from a clinically annotated, publicly available dataset of 267 hormone HR+/HER2− breast cancer patients with chemo-endocrine treatment [18] that shares characteristics with the GeparTrio cohort (Additional file 3: Table S2). We evaluated the expression of HLA-A, HLA-B, and HLA-C using the continuous data and defined cut points corresponding to the observed frequencies of high vs. low HLA class I expression in the IHC dataset (Table 2, Fig. 3). HLA-A, HLA-B, and HLA-C were strongly correlated (Additional file 2: Figure S2). HLA-A, but not HLA-B or HLA-C, was associated with longer distant recurrence-free survival but not with response to therapy (Table 2, Fig. 3).

The association of HLA-A expression with previously described immune-cell-specific metagenes [16] was evaluated in the microarray-based dataset to better understand the association with the immunological tumor infiltrate (Additional file 3: Table S4). Within the HR+/HER− subset, positive correlations of the HLA metagene were found with all signatures (Additional file 3: Table S4). The strongest associations were detected for the T cell signatures (ρ_P = 0.553), myeloid dendritic cells (ρ_P = 0.488), and cytotoxic lymphocyte signatures (ρ_P = 0.471).

Univariate Cox and logistic regression were performed to evaluate the predictive and prognostic influence of the different immune cell populations in HR+/HER2− breast cancer. The metagene representing neutrophils was associated with reduced response to neoadjuvant chemotherapy, while the metagene for the monocytic lineage was associated with better response (Fig. 4). The HLA-A metagene, the monocytic lineage, and the B lineage were associated with higher risk of distant relapse.

To evaluate subtyping effects of HLA-A expression, we correlated the expression with estrogen receptor 1 (ESR1) and aurora kinase A (AUKRA) as a marker of proliferation [20]. There was no strong association of HLA-A with either gene (ρ_P = − 0.164 and − 0.122, respectively; Additional file 3: Table S4).