Introduction
The proline-rich protein 11 (PRR11) gene is located
on human chromosome 17q22-23 and comprises 10 exons and nine
introns (1,2). PRR11 may serve a role in
carcinogenesis across various cancer types (3–6). PRR11
may contribute to prognosis and treatment outcomes in solid tumors,
such as breast cancer, colon cancer, gastric cancer, hepatocellular
carcinoma, cholangiocellular carcinoma, pancreatic cancer, lung
cancer, ovarian cancer and osteosarcoma (7). In numerous malignancies, cell cycle
defects are considered to be key to carcinogenesis. Ji et al
(2) reported that PRR11 expression
increases from the G1 phase to the G2/M phase
of the cell cycle. PRR11 overexpression may contribute to cell
proliferation. Marked inhibition of cell proliferation was also
detected after PRR11 depletion. This outcome suggests that PRR11
may serve a role in the cell cycle, particularly in S-phase
progression and subsequent cell cycle steps (2).
Breast cancer remains the most commonly diagnosed
cancer among women worldwide, with ~2.3 million women diagnosed and
670,000 mortalities globally in 2022 (ref). Breast cancer is a
biologically heterogeneous disease, and therapeutic strategies are
increasingly tailored to distinct molecular subtypes, ranging from
immunotherapeutic approaches under investigation in triple-negative
disease to HER2-directed antibody-drug conjugates in HER2-low
disease (8). Luminal-like breast
cancer is defined as HER2-negative or HER2-positive hormone
receptor-positive breast cancer. Previously, HER2-negative disease
has been defined as disease with a CerbB2 immunohistochemistry
(IHC) score of 0 or an IHC score of 1+ or 2+ without HER2
amplification based on in situ hybridization (ISH) (7). However, it is well-established that
IHC score 1 or score 2+ and no HER2 amplification based on ISH are
distinct entities and are referred to as HER2-low disease (7). HER2-low status is prognostic and
predictive for treatment response for novel therapeutics, such as
trastuzumab deruxtecan, an antibody-drug conjugate targeting HER2
(9). However, there are numerous
gray areas in understanding and addressing treatment sensitivity
and/or resistance in these patients. The prognostic value of PRR11
expression has not been well-documented in patients with estrogen
receptor (ER)+/HER2-low breast cancer, representing an
important gap in the existing literature that the present study
aims to address. ER+/HER2-low breast cancer represents a
large and clinically important luminal subgroup whose therapeutic
landscape has expanded with the introduction of HER2-directed
antibody-drug conjugates (9).
Despite this, the molecular determinants of endocrine response and
resistance within this specific subtype remain incompletely
characterized. Given its established mechanistic link to PI3K
signaling and antiestrogen resistance in ER-positive disease, PRR11
represents a biologically plausible candidate marker in this
setting, providing the rationale for the present exploratory
investigation.
The present study aimed to investigate PRR11
expression and its effects on prognosis in patients with localized
or locally advanced-stage (non-metastatic), ER+ and
HER2-low breast cancer.
Materials and methods
Study design
A total of 124 patients with non-metastatic
ER+/HER2-low breast cancer followed up at Dr.
Abdurrahman Yurtaslan Ankara Oncology Training and Research
Hospital (Ankara, Turkey) between January 2010 and December 2019
were analyzed retrospectively in February 2024. ER-positive (ER,
≥1%) and HER2-low patients were included. The cut-off level for ER
positivity was 1%. For the primary grouping analysis, patients were
categorized into three ER subgroups: ER, 1–50%; ER, 51–89%; and ER,
≥90%. For the detailed PRR11 expression analysis, patients were
additionally analyzed in 10% ER intervals to examine the gradient
relationship between the ER positivity rate and PRR11 expression
levels. The 10% ER interval analysis was adopted to enable a
fine-grained, gradient assessment of the relationship between ER
positivity and PRR11 expression. ER expression is reported as a
continuous percentage (1–100%) in routine pathology, and
accumulating evidence indicates that ER positivity behaves as a
biological continuum rather than a dichotomous variable, with
low-ER (1–10%) tumors being molecularly and clinically distinct
from high-ER tumors (10,11). Decile-based stratification therefore
permits the detection of dose-response patterns that coarser
categorical grouping may obscure. HER2-low status was defined as a
CerbB2 IHC score of 1+ or a CerbB2 IHC score of 2+ without HER2
amplification based on silver ISH (SISH). For analysis, HER2-low
patients were further divided into the CerbB2 IHC score 1+ subgroup
and the CerbB2 IHC score 2+ and SISH-negative subgroup.
Demographic characteristics of the patients such as
age, sex, menopausal status and comorbidities, pathological
characteristics such as pathological subtype, grade, Ki-67 value,
ER percentage, progesterone receptor (PR) percentage and CerbB2 IHC
score, and clinical characteristics such as the treatment
modalities and survival outcomes for disease-free survival (DFS)
and overall survival (OS) were recorded. DFS was defined as the
interval between the diagnosis date and the date of recurrence or
mortality. OS was defined as the interval between diagnosis and
mortality or the last known date of being alive.
IHC
ER and HER2 expression was evaluated by IHC using
formalin-fixed, paraffin-embedded (FFPE) tissue sections. All
specimens were fixed for 24–72 h in room temperature in neutral
buffered 10% formalin. Sections at 4 µm thick were prepared on
positive charged slides. Immunohistochemical staining was performed
on an automated BenchMark ULTRA IHC/ISH platform. After
conditioning in 95°C for 8 min with Cell Conditioner #1, prediluted
PATHWAY anti-HER2/neu (4B5) Rabbit Monoclonal Primary Antibody
(cat. no. 05278368001; Roche Diagnostics) was incubated in 36°C for
36 min. A secondary UltraView Universal DAB Detection Kit (cat. no.
05269806001; Roche Diagnostics) was applied. Hematoxylin
counterstain was applied at 36°C for 16 min and Bluing reagent at
36°C for 4 min. Stained slides were imaged using a Nikon Optiphot 2
microscope. ER positivity was defined as nuclear staining in ≥1% of
tumor cells, scored as a percentage (1–100%). HER2 expression was
scored according to the 2018 American Society of Clinical
Oncology/College of American Pathologists guidelines (7): IHC score 0, no staining or incomplete
faint membrane staining in ≤10% of tumor cells; IHC score 1+,
incomplete faint/barely perceptible membrane staining in >10% of
tumor cells; IHC score 2+, weak to moderate complete membrane
staining in >10% of tumor cells; or IHC score 3+, strong
complete membrane staining in >10% of tumor cells. HER2-low
status was defined as an IHC score of 1+ or an IHC score of 2+ with
negative SISH.
RNA extraction, cDNA synthesis and
reverse transcription-quantitative PCR (RT-qPCR)
Total RNA was obtained from FFPE tumor tissue blocks
using the DiaRex® Total RNA Extraction kit (cat. no.
TR-0877; Diagen İnsan Sağlığı). Briefly, after 5–30 mg of tissue
was homogenized in a 1.5-ml homogenization tube, extraction was
performed according to the kit manufacturer's instructions, and
finally, 30–50 µl total RNA was obtained. Total RNA was stored at
−80°C until analysis.
Preserved samples were thawed on ice, and RNA
concentrations were measured spectrophotometrically (Colibri;
Berthold Technologies GmbH & Co.) to standardize RNA input
prior to cDNA synthesis. cDNA synthesis was performed using the
SOLIScript® RT cDNA Synthesis Kit (Solis BioDyne)
according to the manufacturer's protocol. RT was performed at 50°C
for 5 min, followed by enzyme inactivation at 85°C for 5 min. cDNA
products were stored at −20°C until use.
PRR11 mRNA expression levels were quantified using
the 2−ΔΔCq method (12),
with β-actin (ACTB) serving as the endogenous reference gene for
normalization. β-actin (ACTB) was selected as the endogenous
reference gene because it is a constitutively expressed
housekeeping gene that is widely used and validated for the
normalization of qPCR data in breast tumor tissue, including FFPE
material, and displays acceptable expression stability across
breast cancer specimens, including estrogen receptor-positive
tumors (13). The present study
acknowledges that the use of a single reference gene, rather than a
panel of validated controls, represents a methodological
limitation. SolisFAST® SolisGreen® qPCR Mix
(no ROX), 5X (Solis BioDyne) was used. Each 20 µl reaction
consisted of 4 µl master mix, 5 µl primer mix (containing 0.3 mM
forward and reverse primers), 6 µl distilled water and 5 µl cDNA.
Amplification was performed using a RT-qPCR system (BioRAD CFX-96;
Bio-Rad Laboratories, Inc.) with the following protocol: Initial
denaturation at 95°C for 5 min, followed by 45 cycles of 95°C for 5
sec and 57°C for 30 sec. The primer sequences were as follows:
PRR11 forward, 5′-GAGTCGGTATTTCTTCAAT-3′ and reverse,
5′-CTAAACTCTGGGTTATGC-3′; and ACTB forward,
5′-TGAAGATCAAGATCATTGCT-3′ and reverse, 5′-ATACTCCTGCTTGCTGAT-3′.
Expression values were reported as the fold change relative to the
cohort median (median fold change) and patients were categorized as
having high or low PRR11 expression based on this median threshold.
Relative mRNA expression levels were calculated using the
2−ΔΔCq method using an R package (version 4.3.1; R
Foundation for Statistical Computing; qpcrtools 1.0.1; ggpubr
0.6.0; dplyr 1.1.4; tidyverse 2.0.0; car 3.1–2).
Statistical analysis
Descriptive statistics were used to examine the
demographic and clinicopathological characteristics of the
patients. Categorical variables were compared using the
χ2 test; when >20% of cells had expected counts below
5, Fisher's exact test (Freeman-Halton extension) was used instead.
The unpaired Student's t-test was used to compare two groups when
the data were normally distributed.
The median follow-up duration was calculated using
the reverse Kaplan-Meier method. Survival analyses of 5-year DFS
and 5-year OS were performed using the Kaplan-Meier method and
possible prognostic factors were compared by log-rank test.
Patients were stratified into high PRR11 expression (above median
fold change) and low PRR11 expression (at or below median fold
change) groups for survival analyses. Receiver operating
characteristic (ROC) analysis was performed to evaluate the
discriminatory ability of PRR11 expression for DFS and OS outcomes,
with optimal cut-off values determined using the Youden index. DFS
was defined as the time from the date of diagnosis to the date of
recurrence, if any, or the date of last follow-up. OS was defined
as the time from the date of diagnosis to the date of exitus or
last follow-up. All statistical analyses were performed using SPSS
version 21.0 (IBM Corp.). P<0.05 was considered to indicate a
statistically significant difference.
Results
Baseline characteristics of the
patients
The mean age was 53.4 (±10.5) years. All patients
were female. Of the patients, 51 (41.1%) were premenopausal and 73
(58.9%) were postmenopausal. In terms of pathological features, all
patients had ER+/HER2-low breast cancer. A total of 14
patients (11.3%) had grade 1 breast cancer, 60 patients (48.4%) had
grade 2 breast cancer and 46 patients (37.1%) had grade 3 breast
cancer. The mean Ki-67 value was 29.3% (±17.4%). A total of 66
patients (53.2%) had a CerbB2 IHC score of 1+, while 58 patients
(46.8%) had a score of 2+. The clinical and pathological
characteristics are presented in Table
I.
 | Table I.Baseline characteristics of
patients. |
Table I.
Baseline characteristics of
patients.
| Characteristic | No. of individuals
(n=124) |
|---|
| Mean age, years
(±SD) | 53.4 (±10.5) |
| Sex, n |
|
|
Female | 124 |
| Male | 0 |
| Menopausal status, n
(%) |
|
|
Premenopausal | 51 (41.1) |
|
Postmenopausal | 73 (58.9) |
| Hypertension, n
(%) | 36 (29) |
| Diabetes mellitus,
n (%) | 22 (17.7) |
| Heart disease, n
(%) | 6 (4.8) |
| Lung disease, n
(%) | 3 (2.4) |
| Pathological
subtype, n (%) |
|
|
IDC | 69 (55.6) |
|
ILC | 7 (5.6) |
|
Others | 48 (38.7) |
| Grade, n (%) |
|
| 1 | 14 (11.3) |
| 2 | 60 (48.4) |
| 3 | 46 (37.1) |
| Mean Ki67, %
(±SD) | 29.3 (±17.4) |
| Hormone receptors,
% (±SD) |
|
| ER | 87 (±21.1) |
| PR | 50.8 (±33.4) |
| CerbB2 score
(IHC) |
|
| Score 1
(1+) | 66 (53.2) |
| Score 2
(2+, SISH negative) | 58 (46.8) |
| Stage, n (%) |
|
| 1A | 26 (21) |
| 1B | 1 (0.8) |
| 2A | 37 (29.8) |
| 2B | 37 (29.8) |
| 3A | 12 (9.7) |
| 3B | 1 (0.8) |
| 3C | 10 (8.1) |
The patients were divided into groups based on their
CerbB2 scores. Demographic characteristics, including age, sex and
comorbidities, and pathological features, including
histopathological subtype, grade, Ki-67 value, ER percentage, PR
percentage and stage, were compared between the groups of patients
based on CerbB2 scores. These two groups had similar demographic
and clinicopathological characteristics (Table II).
| Table II.Baseline patients' characteristics
according to CerbB2 IHC score. |
Table II.
Baseline patients' characteristics
according to CerbB2 IHC score.
| Characteristic | CerbB2 score 1
Total (n=66) | CerbB2 score 2
Total (n=58) | P-value |
|---|
| Age, years
(±SD) | 53.4 (±10.5) | 53.4 (±10.6) | 0.148 |
| Sex F/M (n) |
|
| Not applicable |
|
Female | 66 | 58 |
|
|
Male | 0 | 0 |
| Menopausal status,
n (%) |
| 0.250 |
|
Premenopausal | 24 (36.4) | 27 (46.6%) |
|
|
Postmenopausal | 42 (63.6) | 31 (53.4%) |
|
| Hypertension, n
(%) | 19 (28.8) | 17 (29.3%) | 0.949 |
| Diabetes mellitus,
n (%) | 11 (16.7) | 11 (19%) | 0.738 |
| Heart disease, n
(%) | 5 (7.6) | 1 (1.7%) | 0.130 |
| Lung disease, n
(%) | 2 (3%) | 1 (1.7%) | 0.637 |
| Pathological
subtype, n (%) |
|
| 0.957 |
|
IDC | 36 (54.5%) | 33 (56.9%) |
|
|
ILC | 4 (6.1%) | 3 (5.2%) |
|
Others | 26 (39.4%) | 22 (37.9%) |
| Grade, n (%) |
|
| 0.304 |
| 1 | 7 (10.6%) | 7 (12.1%) |
|
| 2 | 29 (43.9%) | 31 (53.4%) |
| 3 | 29 (43.9%) | 17 (29.3%) |
| Ki67, % (±SD) | 30.9 (±19.7) | 27.5 (14.3%) | 0.247 |
| Hormone receptors,
% (±SD) |
|
| ER | 85.3 (±22.9) | 88.9 (±19) | 0.595 |
| PR | 53.3 (±31.7) | 48 (±35.2) | 0.569 |
| Stage, n (%) |
| 0.716 |
| 1A | 14 (21.2%) | 12 (20.7%) |
|
| 1B | 0 | 1 (1.7%) |
| 2A | 21 (31.8%) | 16 (27.6%) |
| 2B | 17 (25.8%) | 20 (34.5%) |
| 3A | 6 (9.1%) | 6 (10.3%) |
| 3B | 1 (1.5%) | 0 |
| 3C | 7 (10.6%) | 3 (5.2%) |
The patients were also divided into three subgroups
based on the ER percentage: ER, 1–50% (n=11); ER, 51–89% (n=47) and
ER ≥90% (n=66). The relationship between ER distributions and
CerbB2 scores was examined. A total of 8 patients (12.1%) with a
CerbB2 IHC score of 1+ were in the ER 1–50% group, 24 (36.4%) were
in the ER 51–89% group and 34 (51.5%) were in the ER >90% group.
Among the patients with a CerbB2 IHC score of 2+ (SISH-negative), 3
(5.2%) patients were in the ER 1–50% group, 23 (39.7%) in the ER
51–89% group and 32 (55.2%) in the ER >90% group. There was no
difference between the patients with a CerbB2 IHC score of 1+ and
2+ in terms of ER groups (P=0.404).
A total of 96 (77.4%) patients received adjuvant
chemotherapy, 14 patients (11.3%) received neoadjuvant chemotherapy
(NAC) and 14 patients (11.3%) received adjuvant endocrine treatment
only (no chemotherapy). The pathological complete response (pCR)
rate was 21.4% in 3 of 14 patients who received neoadjuvant
treatment. According to the residual cancer burden (RCB) index
(14), the neoadjuvant treatment
response in 6 patients was classified as partial response (RCB-II).
A total of 5 patients were resistant to neoadjuvant treatment
(RCB-III). While 87 patients (70.2%) received adjuvant
radiotherapy, 37 patients (29.8%) did not. All patients received
adjuvant endocrine therapy, including tamoxifen or an aromatase
inhibitor, plus a luteinizing hormone-releasing hormone analog,
based on their menopausal status and comorbidities.
PRR11 expression analysis
PRR11 expression status was analyzed in relation to
the percentage of ER and CerbB2 IHC scores. When evaluating PRR11
expression by ER percentage, patients were grouped into 10% ER
intervals. The median PRR11 fold change in each subgroup was as
follows: 0.31 in the ER 1–10% subgroup, 0.50 in the ER 20–30%
subgroup, 1.19 in the ER 40–50% subgroup, 1.23 in the ER 70–80%
subgroup, 1.41 in the ER 80–90% subgroup and 1.55 in the ER >90%
subgroup (Fig. 1). A direct
proportional relationship was observed between the ER positivity
rate and PRR11 expression, particularly when the ER level was
>40%.
PRR11 expression was also evaluated in two subgroups
based on CerbB2 IHC scores: IHC score 1+ and IHC score 2+
(ISH-negative). While the median fold change for PRR11 was 1.717 in
the CerbB2 IHC score 1+ subgroup, it was 0.999 in the CerbB2 IHC
score 2+ subgroup (P=0.39; Fig.
2).
PRR11 expression was also evaluated in the tissues
at diagnosis and in the postoperative residual tumors of 11
patients who did not achieve pCR with neoadjuvant treatment. After
neoadjuvant treatment, PRR11 expression was decreased in 4 patients
(36%) and increased in 7 patients (64%; Table III). However, exploratory paired
analysis showed no statistically significant difference in median
PRR11 fold change before and after neoadjuvant treatment (median,
2.318 vs. 1.540; Wilcoxon signed-rank test, P=0.465).
| Table III.PRR11 expression before and after
neoadjuvant treatment. |
Table III.
PRR11 expression before and after
neoadjuvant treatment.
|
| Median PRR11 fold
change |
|---|
|
|
|
|---|
| Patient no. | Before neoadjuvant
treatment | After neoadjuvant
treatment |
|---|
| 1 | 0.248 | 0,077 |
| 2 | 0.112 | 0.162 |
| 3 | 2.318 | 1.417 |
| 4 | 0.234 | 0.560 |
| 5 | 0.295 | 1.540 |
| 6 | 0.438 | 1.067 |
| 7 | 6.161 | 5.553 |
| 8 | 4.136 | 6.956 |
| 9 | 5.630 | 6.719 |
| 10 | 2.645 | 2.965 |
| 11 | 5.457 | 2.845 |
Survival analysis
The 5-year DFS and 5-year OS of the patients were
evaluated. Survival analyses were performed to examine potential
prognostic factors affecting survival outcomes, including age,
menopausal status, pathological subtype, grade, Ki-67 value, ER
percentage range, CerbB2 IHC score and stage at diagnosis. None of
these factors significantly affected 5-year DFS and 5-year OS in
the univariate analysis (Table
IV).
| Table IV.Univariate analysis of possible
prognostic factors for survival. |
Table IV.
Univariate analysis of possible
prognostic factors for survival.
| Characteristic | 5-year DFS, % | Median DFS (95%
CI) | P-value | 5-year OS, % | Median OS (95%
CI) | P-value |
|---|
| Age, years |
| 0.879 |
| 0.091 |
|
<55 | 91.1 | NR |
| 100 | NR |
|
|
≥55 | 92.5 | NR | 97.7 | NR |
|
| Menopausal
status |
| 0.515 |
| 0.171 |
|
Premenopausal | 89 | NR |
| 100 | NR |
|
|
Postmenopausal | 93.8 | NR | 98.1 | NR |
| Ki67, % |
| 0.240 |
| 0.809 |
|
<20 | 87.3 | NR |
| 97.3 | NR |
|
|
≥20 | 96 | NR | 100 | NR |
| CerbB2 IHC
score |
| 0.317 |
| 0.955 |
| +1 | 92.3 | NR |
| 98 | NR |
|
| +2 | 91 | NR | 100 | NR |
| Grade |
| 0.912 |
| 0.291 |
| Grade 1
and 2 | 92.3 | NR |
| 98.3 | NR |
|
| Grade
3 | 90 | NR | 100 | NR |
| ER range, % |
| 0.213 |
| 0.275 |
|
1-50 | 70 | NR |
| 100 | NR |
|
|
51-89 | 93 | NR | 100 | NR |
|
≥90 | 94.6 | NR | 97.8 | NR |
| Stage |
| 0.162 |
| 0.734 |
| 1 | 100 | NR |
| 100 | NR |
|
| 2A | 88.1 | NR | 96.3 | NR |
| 2B | 94.4 | NR | 100 | NR |
| 3 | 81.4 | NR | 100 | NR |
| Histopathological
subtype |
| 0.934 |
| 0.608 |
|
IDC | 92.7 | NR |
| 98.1 | NR |
|
|
ILC | 85.7 | NR | 100 | NR |
The 5-year DFS rate, evaluated in groups, based on
ER percentage ranges, was 70% in the ER 1–50% subgroup, 93% in the
ER 51–89% subgroup and 94.6% in the ER >90% subgroup (P=0.213).
There was no significant difference in 5-year DFS between groups
based on the CerbB2 IHC score (92.3% for the CerbB2 IHC score 1+
subgroup vs. 91% for the CerbB2 IHC score 2+ subgroup; P=0.317)
(Table IV).
Survival analyses were also performed to compare
patient outcomes according to PRR11 expression levels. Exploratory
ROC analysis showed that PRR11 expression had no meaningful
discriminatory ability for DFS (AUC=0.409). ROC analysis for OS was
not considered reliable because of the limited number of events in
this cohort (n=2), precluding robust curve estimation (data not
shown). Patients were stratified into two groups based on the
median PRR11 fold change: High PRR11 expression (above the median)
and low PRR11 expression (at or below the median). Kaplan-Meier
analysis revealed no statistically significant difference between
the two groups in terms of 5-year DFS (P=0.272; data not shown).
Formal log-rank testing for OS was not performed due to the
insufficient number of events (n=2). These results indicated that
PRR11 expression, as assessed in the present cohort, was not
independently associated with survival outcomes in early-stage
ER+/HER2-low breast cancer.
Discussion
PRR11 is expressed at low levels in normal tissues
(15). PRR11 expression may serve a
role in the cell cycle, particularly during the G2/M
phase, as well as in cell filopodia formation and motility
(2,16). Therefore, PRR11 expression is being
investigated as a potential prognostic marker and treatment target
across numerous cancer types. Zhang et al (17) evaluated PRR11 expression levels in
33 different tumor types using The Cancer Genome Atlas (TCGA). The
authors reported that PRR11 expression was upregulated in 19 cancer
types, including breast cancer, and that PRR11 upregulation might
be a poor prognostic factor in 10 cancer types but not in breast
cancer. Wang et al (5) also
analyzed RNA-sequencing data from TCGA to evaluate the prognostic
significance of PRR11-spindle and kinetochore associated complex
subunit 2 (SKA2) gene pair expression and p53 expression in breast
cancer. The authors determined that the expression of the
PRR11-SKA2 gene pair was lower in patients with breast cancer
without p53 mutation when compared with those with p53 mutation,
leading to improved DFS (P<0.0001) (5).
Breast cancer is a heterogeneous histopathological
process with various pathological and molecular features. The
prognostic value of PRR11 expression is not well-documented in
patients with ER+/HER2-low breast cancer. In the present
study, PRR11 expression levels were analyzed according to ER levels
and CerbB2 IHC scores in patients with ER+/HER2-low
early-stage (non-metastatic) breast cancer. A positive association
was observed between PRR11 expression and the ER percentage.
Notably, patients with higher ER percentages had higher PRR11
expression, since higher ER levels are a favorable prognostic
factor in luminal-like breast cancer, while higher PRR11 expression
is a poor prognostic factor based on the literature (5,18).
Therefore, the coincidence of higher PRR11 expression and higher ER
percentages presents a dilemma and requires further
investigation.
One potential explanation for this positive
association is that PRR11 may function, at least in part, as an
ER-regulated gene. If PRR11 transcription is driven by ER
signaling, its elevated expression in highly ER-positive tumors may
not necessarily reflect aggressive tumor biology in the same manner
as observed in ER-negative malignancies such as lung or gastric
cancer. Although a formal analysis of ER-binding motifs within the
PRR11 promoter region was beyond the scope of the present study,
this hypothesis is biologically plausible given the known role of
ER as a transcriptional activator (19). A study by Lee et al (1) demonstrated that PRR11 overexpression
amplified PI3K signaling and promoted antiestrogen resistance in
ER-positive breast cancer, suggesting a bidirectional relationship
between ER activity and PRR11 expression. Future studies
incorporating chromatin immunoprecipitation or promoter analysis
are warranted to determine whether PRR11 is a direct
transcriptional target of ER signaling.
HER2-low breast cancer is a novel entity with
prognostic and predictive value for anti-HER2 antibody-drug
conjugates, such as trastuzumab deruxtecan. There are increasing
data regarding differences in clinicopathological features and
prognosis between patients with HER2-low and HER2-negative (CerbB2
score 0) breast cancer (20–22).
To the best of our knowledge, there are no clear data on the
relationship between PRR11 expression and HER2 status in breast
cancer. In the present study, patients with a CerbB2 IHC score of
1+ had higher PRR11 expression when compared with patients with a
CerbB2 IHC score of 2+, although this difference did not reach
statistical significance (P=0.39). This trend, if significant in
larger cohorts, could suggest that HER2-low breast cancer is not a
biologically uniform entity and that IHC-based subcategorization
may carry molecular relevance. The sample size limitation for this
specific comparison (n=66 vs. n=58) is acknowledged, and larger
cohorts are needed to determine whether this trend is reproducible.
The limitation of relying solely on IHC/SISH for HER2-low
classification, without correlation with HER2 mRNA levels, is also
acknowledged as a direction for future research.
PRR11 expression levels did not exhibit a
statistically significant association with 5-year DFS or OS in the
present cohort. This finding contrasts with the reported prognostic
significance of PRR11 in other malignancies, including lung,
gastric and pancreatic cancer (4,6,23,24).
Several factors may account for this discrepancy. First, the
uniform administration of adjuvant endocrine therapy across the
entire cohort may have masked the independent prognostic
contribution of PRR11. Second, the relatively limited follow-up
duration and the low event rate inherent to an early-stage patient
population may have reduced the statistical power to detect a
survival difference. Third, as aforementioned, PRR11 expression in
highly ER-positive tumors may reflect ER-driven transcriptional
activity rather than intrinsic oncogenic aggressiveness,
attenuating its prognostic impact in this specific subtype.
Furthermore, the exceptionally low mortality rate observed in this
cohort (only 2 mortalities among 124 patients) reflects the
favorable OS characteristic of early-stage ER+/HER2-low
breast cancer treated with standard multimodal therapy (25,26).
While this represents a clinically favorable outcome, it
substantially limits the statistical power to detect any survival
difference attributable to PRR11 expression. It is therefore
plausible that the OS of this patient population may have masked a
potential prognostic effect of PRR11, should one exist. Larger
cohorts with longer follow-up and higher event rates will be
necessary to address this question adequately. A further limitation
concerns the statistical modeling. Owing to the low number of
events in this early-stage cohort (13 DSF events and 2 mortalities
among 124 patients), a multivariable Cox proportional-hazards model
could not be reliably constructed, as the resulting
events-per-variable ratio would fall well below accepted thresholds
for stable estimation. Consequently, the independent prognostic
contribution of PRR11, adjusted for established clinicopathological
covariates, could not be determined, and the present survival
findings should be interpreted as univariate and exploratory.
PRR11 expression may also carry potential predictive
value for treatment outcomes. Lee et al (1) reported an association between PRR11
overexpression and antiestrogen resistance via the PI3K signaling
pathway in ER-positive breast cancer. In the present study, PRR11
expression was evaluated in residual tumor tissues from 11 patients
who did not achieve a pCR following NAC. Post-treatment PRR11
expression levels were higher than pre-treatment levels in 7 of
these 11 patients (64%). While this observation is intriguing and
raises the hypothesis that elevated PRR11 expression in residual
disease may reflect chemotherapy-resistant tumor clones or an
adaptation mechanism contributing to treatment resistance, these
findings must be interpreted with considerable caution. The sample
size of 11 patients is insufficient to draw statistically
meaningful conclusions, and no formal statistical comparison was
performed for this subgroup. These results should therefore be
regarded strictly as preliminary, hypothesis-generating
observations. Larger prospective cohorts with standardized NAC
protocols and systematic pre- and post-treatment tissue sampling
are needed to validate whether PRR11 expression has genuine
predictive value for NAC response in ER+/HER2-low breast
cancer.
Besides its potential prognostic and predictive
value, PRR11 expression may also serve as a diagnostic marker for
breast cancer. In a study evaluating PRR11 expression in fresh
biopsies of both tumoral and adjacent non-tumoral tissues in 70
patients with breast cancer, PRR11 upregulation could distinguish
invasive breast cancer tissue from normal tissue with a sensitivity
of 92.86%, specificity of 85.71%, AUC of 0.916 and Youden index of
78.6% (27). Additionally, the
authors noted an association between PRR11 upregulation and ER/PR
positivity, which supports the positive association between ER
levels and PRR11 expression observed in the present study.
PRR11 expression is also a potential therapeutic
target in other solid tumors, such as non-small cell lung cancer
(NSCLC). Zhao (18) reported that
targeted depletion of PRR11 expression in lung cancer cells led to
cell cycle arrest and apoptotic cell death in patients with NSCLC.
In another study, silencing of PRR11 was shown to stimulate
autophagy (28). The AKT/mTOR
pathway is considered to be one of the primary pathways regulating
autophagy (29,30). Inhibition of PRR11 expression may
indirectly stimulate autophagy via the AKT/mTOR pathway. The
PI3K/AKT/mTOR pathway is a signaling pathway in which gene
alterations, such as phosphatidylinositol-4,5-bisphosphate 3-kinase
catalytic subunit α mutation, occur during carcinogenesis and
follow-up (31). The potential
interaction between the PI3K/AKT/mTOR signaling pathway and PRR11
upregulation should therefore also be evaluated to determine
potential targets in breast cancer.
In conclusion, the present study revealed a positive
association between PRR11 expression and the ER positivity rate in
ER+/HER2-low early-stage breast cancer. PRR11 expression
did not exhibit a statistically significant association with 5-year
DFS or OS in the present cohort, likely reflecting the favorable OS
of this early-stage population and the confounding effect of
uniform adjuvant endocrine therapy. Higher PRR11 levels in residual
disease after NAC represent a preliminary, hypothesis-generating
observation regarding its potential predictive value. PRR11
expression differed according to CerbB2 IHC score in the HER2-low
population. Randomized clinical trials and larger prospective
studies are needed in this area.
Acknowledgements
Not applicable.
Funding
This work was supported by the Turkish Society of Medical
Oncology.
Availability of data and materials
The data generated in the present study may be
requested from the corresponding author.
Authors' contributions
MD and TB were supervisors. SCI and NB prepared
patient pathology specimens from the pathology laboratory archives
for analysis. HBE and TB performed the necessary tests for PRR11
expression. AT collected and analyzed the data and wrote the
manuscript. MD consulted on the concept analyses during design of
the research strategy and writing of the manuscript. MD reviewed
and edited the manuscript. AT and TB confirm the authenticity of
all the raw data. All authors read and approved the final version
of the manuscript.
Ethics approval and consent to
participate
The present study was approved by the Clinical
Research Ethics Committee of Dr. Abdurrahman Yurtaslan Ankara
Oncology Training and Research Hospital, Ankara, Turkey (approval
no. 2022-12/2195), and was conducted in accordance with the
principles of the Declaration of Helsinki. As this was a
retrospective study based on archived FFPE tissue samples, the
requirement for written informed consent was waived by the ethics
committee, and all patient data were anonymized prior to
analysis.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
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