FEATURES AND PROGNOSIS OF STAGES I–IV BREAST CANCER SUBTYPES AT THE CLINICAL HOSPITAL OF BOTUCATU MEDICAL SCHOOL – UNESP

Abstract

Objective: This study is proposed to assess the survival of patients with breast cancer according to the molecular
classifications estimated by immunohistochemistry (IHQ) and to evaluate the importance of Ki-67 proliferation index
in the distinction of luminal subgroups and as a prognostic factor for Brazilian women with breast cancer in clinical
stages I–IV. Methods: This is a retrospective, observational cohort study that included 842 patients with invasive breast cancer diagnosed between 2009 and 2016 who were treated at the Clinical Hospital of Botucatu Medical School –
UNESP. Data collection was performed from June 2020 to May 2021 with the observation of a minimum survival time
of 5 years and a maximum of 12 years. The study was approved by the institution’s ethics committee. Inclusion criteria
were women with at least 18 years of age who were diagnosed with invasive breast carcinoma registered as new cases
in stages I–IV between 2009 and 2016 and whose treatment was initiated at the Clinical Hospital of Botucatu Medical
School – UNESP. Data were collected from electronic medical records and inserted into an Excel spreadsheet. After this
evaluation, breast cancer was categorized into five molecular subtypes based on immunohistochemical profiles according to the following classification:
1- Luminal Ki-67 >10% Classification
“Luminal A”: ER positive, PR positive, HER2 negative and Ki-67 ≤10%
“Luminal B”: ER positive, PR positive or negative, HER2 negative and Ki-67 >10%
“Luminal B-HER2 positive (hybrid): ER positive, PR positive or negative, HER2 positive, any Ki-67 index
“HER2-enriched”: ER negative, PR negative and HER2 positive with any Ki-67 index
“Triple negative”: ER negative, PR negative, HER2 negative with any Ki-67 index
2- Luminal Ki-67 >20% Classification
“Luminal A”: ER positive, PR positive, HER2 negative and Ki-67 ≤20%
“Luminal B”: ER positive, PR positive or negative, HER2 negative and Ki-67 >20%
“Luminal B-HER2 positive (hybrid): ER positive, PR positive or negative, HER2 positive, any Ki-67 index
“HER2-enriched”: ER negative, PR negative and HER2 positive with any Ki-67 index
“Triple negative”: ER negative, PR negative, HER2 negative with any Ki-67 index
Another stratification using only Ki-67 index as reference was performed:
3- Ki-67 >10% Classification
Group 1: Ki-67 ≤10%
Group 2: Ki-67 >10%

After this evaluation, breast cancer was categorized into five molecular subtypes based on immunohistochemical profiles according to the following classification:
1- Luminal Ki-67 >10% Classification
“Luminal A”: ER positive, PR positive, HER2 negative and Ki-67 ≤10%
“Luminal B”: ER positive, PR positive or negative, HER2 negative and Ki-67 >10%
“Luminal B-HER2 positive (hybrid): ER positive, PR positive or negative, HER2 positive, any Ki-67 index
“HER2-enriched”: ER negative, PR negative and HER2 positive with any Ki-67 index
“Triple negative”: ER negative, PR negative, HER2 negative with any Ki-67 index
2- Luminal Ki-67 >20% Classification
“Luminal A”: ER positive, PR positive, HER2 negative and Ki-67 ≤20%
“Luminal B”: ER positive, PR positive or negative, HER2 negative and Ki-67 >20%
“Luminal B-HER2 positive (hybrid): ER positive, PR positive or negative, HER2 positive, any Ki-67 index
“HER2-enriched”: ER negative, PR negative and HER2 positive with any Ki-67 index
“Triple negative”: ER negative, PR negative, HER2 negative with any Ki-67 index
Another stratification using only Ki-67 index as reference was performed:
3- Ki-67 >10% Classification
Group 1: Ki-67 ≤10%
Group 2: Ki-67 >10%
After this evaluation, breast cancer was categorized into five molecular subtypes based on immunohistochemical profiles according to the following classification:
1- Luminal Ki-67 >10% Classification
“Luminal A”: ER positive, PR positive, HER2 negative and Ki-67 ≤10%
“Luminal B”: ER positive, PR positive or negative, HER2 negative and Ki-67 >10%
“Luminal B-HER2 positive (hybrid): ER positive, PR positive or negative, HER2 positive, any Ki-67 index
“HER2-enriched”: ER negative, PR negative and HER2 positive with any Ki-67 index
“Triple negative”: ER negative, PR negative, HER2 negative with any Ki-67 index
2- Luminal Ki-67 >20% Classification
“Luminal A”: ER positive, PR positive, HER2 negative and Ki-67 ≤20%
“Luminal B”: ER positive, PR positive or negative, HER2 negative and Ki-67 >20%
“Luminal B-HER2 positive (hybrid): ER positive, PR positive or negative, HER2 positive, any Ki-67 index
“HER2-enriched”: ER negative, PR negative and HER2 positive with any Ki-67 index
“Triple negative”: ER negative, PR negative, HER2 negative with any Ki-67 index
Another stratification using only Ki-67 index as reference was performed:
3- Ki-67 >10% Classification

Group 1: Ki-67 ≤10%
Group 2: Ki-67 >10%
4- Ki-67 >20% Classification
Group 1: Ki-67 ≤20%
Group 2: Ki-67 >20%
Statistical analysis
Initially, a descriptive analysis was performed with the calculation of mean and standard deviation for continuous variables and frequencies and percentages for categorized variables. The primary outcome was overall survival (OS) and the
secondary outcome was cancer-specific survival (CSS). OS was defined as the time interval between the date of diagnosis
and the date of death (related to breast cancer or death from any cause). CSS was defined as the time interval between
the date of diagnosis and the date of death related to breast cancer. Follow-up data were collected between October 2020
and May 2021. To calculate survival outcomes, the Kaplan-Meier estimator was used, followed by the log-rank test and
the Sidak test for comparison between groups. A Cox proportional hazard model was used to analyze the association
between different subgroups and survival. The chi-square test was used to study the association of variables and to compare proportions. A level of significance of 5% or the corresponding p-value was adopted in all tests. The analyses were
performed using the Statistical Analysis System 9.4 program. Results: A total of 842 patients with breast cancer were included in the study. Clinical features are shown in Table 1. The mean age was 56.4 years, 35.9% of patients were 50 years or
younger, and 64.3% were postmenopausal. Most patients (60.9%) had only elementary education and 14.3% had a higher
education level. Nulliparous women accounted for 9.5% of cases and 26.1% of patients had a first- or second-degree family
history of breast or ovarian cancer.
Considering the anatomical staging, most cases (63%) were in stage I or II and 6.9% in stage IV. Histological grade 3 was
observed in 39.4% of the cancers and 73.4% of the patients had ER positive tumors, 62.4% PR-positive tumors, and 21.2%
HER2-positive tumors. Luminal (HER2-negative) subtypes accounted for 47.2% of cases, followed by TN (15.2%), Luminal
B-HER2 positive (14.1%), and HER2-enriched (7.3%). As for the Ki-67 proliferation index, in 24.5% of cases it was ≤10%,
and in 42.8% of cases it was >20%. Conservative surgical treatment was performed in 47% of cases and mastectomy with
immediate reconstruction in 13.4% of patients. Most patients underwent systemic chemotherapy (76.9%) and radiation
therapy (82.7%). It was observed that, regardless of the subgroup, most patients were in stage II. With regard to Ki-67
index, 84.9% of patients with Ki-67 ≤10% were in stages I and II and 38.1% of patients with Ki-67 >20% were in stages III
and IV. TN tumors accounted for 47.7% of cases in patients younger than 40 years, 23.4% of tumors between 40 and 50
years, and 15.2% of tumors in patients older than 50 years of age. Luminal subtypes accounted for 30.1% of tumors in
patients younger than 40 years, 45.6% of tumors between 40 and 50 years, and 59.7% of tumors in patient older than 50
years of age. Considering Ki-67, 75.8% of patients younger than 40 years had the index greater than 20% and only 13.4%
had a Ki-67 ≤10%. Between 40 and 50 years, 62.6% of patients had a Ki-67 >20% and 23.1% had a level ≤10%. In patients
older than 50 years, Ki-67 was higher than 20% in 46.4% and ≤10% in 37.8% of cases. Luminal A tumors (regardless of the
Ki-67 cutoff value) had the lowest rates of chemotherapy, in which one-third of patients with this subtype did not undergo
chemotherapy. Patients with triple-negative and luminal B (Ki-67 >20%) tumors were the groups that mostly underwent
systemic cytotoxic treatment (89% of the patients). Patients with a Ki-67 ≤10% did not undergo chemotherapy in one-
-third of cases, whereas 91.8% of patients with Ki-67 >20% underwent systemic treatment. When assessing the type of
surgery according to tumor subtype, mastectomy was more performed in the HER2-enriched group, corresponding to
41.9% of the surgeries performed in this tumor subtype. As for conservative surgery, it was more frequently performed
in the Luminal A (Ki-67 ≤20%) group, corresponding to 58.2% of the cases of surgery in this subtype. The group with the
highest percentage of reconstruction was the Luminal B-HER2 positive, in which the mastectomy followed by reconstruction corresponded to 20.3% of cases. The survival rate was 94.4% in stage I, 85.2% in stage II, 64.7% in stage III, and
19.6% in stage IV. OS was 78.2%. A better survival was observed in the Luminal A subgroup (88.9%), regardless of the Ki-67 cutoff value (10% or 20%), and a worse survival was observed in the triple-negative subgroup (60.5%). In Figures 3 and 4,
survival curves of the patients according to the Ki-67 index are shown. Worse survival rates were observed in the groups
with a higher Ki-67 index, both in those groups with a Ki-67 >10% and >20%. Conclusion: Our study is in accordance with
some findings of the AMAZONA study. A higher percentage of breast cancer diagnoses in the population under 50 years of
age and a lower percentage of initial breast cancer were observed in our study when compared to data from high-income
countries. We also observed that survival was related to staging as shown in previous studies from developed countries.
Another finding was that classification of breast tumors by IHQ reflects different survival curves between Luminal A,
hybrid, HER2-enriched, and TN groups regardless of the Ki-67 level. Although we were unable to establish a cutoff value
that would separate survival rates between luminal groups, Ki-67 had an independent prognostic value, and high values
of this marker were associated with a greater use of chemotherapy.