P16 Expression in Human Breast Carcinoma and its Relationship to Clinicopathological Parameters.

INTRODUCTION
Breast cancer is the most common non-cutaneous malignant tumor and is the second cause of cancer deaths among women.[1] Data reveal an increase in the incidence of this malignancy in Asia and a decrease in the mean age of Iranian patients in recent years.[23] Breast cancer is caused by the accumulation of mutations in drivers and other genes that provide cells a proliferative advantage.[45] The occurrence of genetic abnormalities in the major genes that control cell growth can lead to the onset of carcinogenesis.[6] One of the most common causes of human cancers is the disruption of cell cycle checkpoints. The p16INK4a is a tumor suppressor gene located on chromosome 9p21.[7] p16 protein, the product of that gene, is one of the negative regulators of the cell cycle which causes G1 arrest by blocking the G1 to S transfer in the cell cycle. p16 inhibits cyclin-dependent kinase 4/6 (CDKs 4/60).[8] CDKs 4/6 phosphorylate Retinoblastoma protein (pRb), resulting in pRb inactivation. pRb phosphorylation by CDKs 4/6 with subsequent inactivation of this protein is an important step in cell cycle progression. Deletions and mutations of p16 in human cancer cell lines point to the significant role of p16 in carcinogenesis.[910] With the activation of telomerase, cells may eventually progress to immortality and cancer.[111213] The prognostic impact of p16 alteration has been reported in some human cancers.[1415161718] Based on the functions of p16, it would be expected that p16 overexpression reduces the likelihood of cancer. However, when p16 mutation is associated with abrogation of p16 signaling, compromised Rb signaling will lead to overexpression of mutant p16 in cells, which continue to proliferate and bypass the senescence.[19] The majority of human cancers show dysregulation of p16.[2021] Alterations of p16 expression have been found to be an early event in the transition from premalignant to malignant tumors.[8] Previous studies on p16 expression in breast cancer and its relationship with known prognostic factors are limited. The pattern of p16 expression is variable in breast tumors.[22] Estrogen receptor-negative breast cancers with a loss of p16 expression have been found to be resistant to treatment.[17]
Antibodies to p16 have been found to be increased in the circulation of patients with breast cancer and it has been shown that the levels of IgG antibody to this protein can be a promising marker for early detection of breast carcinoma.[2324] Due to the limited results of previous studies in this field, we aimed to conduct this study to investigate the expression of p16 in invasive breast carcinoma and normal tissue adjacent to the tumor, as well as normal mammoplasty samples. We also investigated the relationship of p16 expression with some prognostic factors of breast cancer.

MATERIALS AND METHODS
We did this study on paraffin tissue blocks. Samples included 40 invasive breast carcinoma (tumor group) and their adjacent normal tissue (tumor control), as well as 30 normal mammoplasty specimens (normal control) from the pathology lab of Al-Zahra Hospital, Isfahan, Iran, from 2016 to 2020. We had study approval from the ethical committee of Isfahan University of Medical Sciences, Isfahan, Iran (Ethical code: IR.MUI.MED.REC 1398-680).
Inclusion criteria for the tumor group were mastectomies or breast lumpectomies with invasive breast carcinoma having normal tissue adjacent to the tumor and dissected axillary lymph nodes. Data concerning age, tumor size, tumor grade, carcinoma subtype, and the number of axillary lymph nodes with metastasis were available from pathology reports. Mammoplasties with normal breast histology were included as the normal control group. Exclusion criteria for the tumor group were tumor specimens lacking normal breast tissue and/or axillary nodes. In the normal control group, mammoplasties showing various breast pathologies were excluded from the study.
We used the easy sampling method in this study. The following formula was used for sample size calculation with a 95% Confidence Interval (CI):

Following sample collection, immunohistochemistry (IHC) was used to stain specimens with p16 antibody (Monoclonal Antibody, Master Diagnostica, Spain). The tissue block of a cervical conization specimen with cervical intraepithelial neoplasia 3 showing p16 block staining was used as a positive control. Areas of the normal squamous epithelium of the same block were used as a negative control. Sections with five-micron thickness were prepared from tissue blocks and stained by p16 antibody as follows:
They were incubated at 37°C in the oven for 48 h, dewaxed by 100% xylol, rehydrated by 100%, 85%, and 75% ethanol, rinsed in 10% phosphate-buffered saline (PBS) solution, incubated for 30 min in 10% H2O2 and methanol for blocking of endogenous peroxidase activity, rinsed in 10% PBS solution, incubated for 14 min in the microwave in citrate-buffered solution (PH = 6.1), rinsed in 10% PBS solution, exposed to blocking serum for 30 min for blocking of endogenous non-specific bindings, dried, exposed to the primary monoclonal antibody of p16 and incubated for 30 min at room temperature, rinsed in 10% PBS solution, exposed to a broad-spectrum secondary antibody for 30 min, exposed to horseradish peroxidase-streptavidin for 30 min, exposed to diaminobenzidine for 10 min, rinsed in 10% PBS solution, dehydrated by 75%, 85%, and 100% ethanol, and counterstained by hematoxylin.

Investigation and analysis of IHC samples
The intensity and percentage of p16 nuclear staining in carcinoma cells and normal breast epithelium were evaluated by a pathologist (supervisor) and a pathology resident according to the scoring system introduced in a previously published report.[25]
Each sample was scored grounded on the maximum intensity of nuclear staining as follows:

0: negative

1: weakly positive

2: moderately positive

3: strongly positive

Each sample was also scored based on the extent of nuclear staining (percentage of stained nuclei) as follows:

0: less than 5%

1: between 5% and 25%

2: between 25% and 50%

3: between 50% and 75%

4: more than 75%

To determine the final score of each sample, the staining intensity score was multiplied by the staining extent score. The lowest and highest final scores were 0 and 12, respectively. The final score was then semi-quantitatively divided into three groups:

Negative: 0 to 4

Weak: 5 to 8

Strong: 9 to 12

We then compared the intensity and extent of p16 nuclear staining between the three groups. We also studied the relationship between p16 nuclear expression and age, tumor size, tumor grade, carcinoma subtype, and lymph node involvement in the tumor group.

Statistical analysis
We used SPSS software, version 16, to analyze data concerning p16 staining, age, tumor size, tumor grade, carcinoma subtype, and the number of involved lymph nodes. A P value less than 0.05 was considered significant. Data were reported as frequency, mean, and standard deviation (SD). Fisher's exact test and Spearman correlation coefficient were used to examine the relationship between p16 expression and prognostic variables in the tumor group.

RESULTS
In this study, we examined 40 specimens of invasive breast carcinoma (tumor group) and normal tissue adjacent to these carcinoma specimens (tumor control group), as well as 30 normal control specimens from mammoplasty surgery (normal control group).
The mean age in the carcinoma group was 47.52 ± 1.52 years with a minimum age of 32 years and maximum age of 76 years. The mean age in the mammoplasty group was 33.97 ± 6.55 years with a minimum age of 23 years and maximum age of 45 years. Data concerning clinicopathological prognostic factors in the carcinoma group have been presented in Table 1.
Data concerning intensity, extent, and final score of p16 nuclear expression in the three groups have been presented in Table 2.
The intensity, extent, and final score of p16 nuclear staining showed a statistically significant difference between the tumor group and the tumor control group. In general, it was found that the intensity, extent, and final score of staining were all higher in the tumor group than the tumor control group [Table 3 and Figures 1-3].
Similarly, the intensity, extent, and final score of p16 nuclear staining showed a statistically significant difference between the tumor group and the normal control group. In general, it was found that the intensity, extent, and final score of staining were all higher in the tumor group than in the normal control group [Table 4].
Intensity, extent, and final score of p16 nuclear staining showed no statistically significant difference between tumor control and normal control groups [Table 5].
In the tumor group, there was a significant direct relationship between the intensity of p16 nuclear staining and the number of involved lymph nodes. No significant relationship was found between tumor size and age and p16 nuclear staining [Table 6].
Tumor grade and carcinoma subtype showed no significant relationship with p16 nuclear staining [Tables 7 and 8].

DISCUSSION
This study was designed to compare p16 nuclear expression in invasive breast carcinoma (tumor group) with normal tissue adjacent to carcinoma (tumor control group) and normal mammoplasty specimens (normal control group). There was a significant difference in intensity, extent, and final score of p16 nuclear expression between the tumor group and normal control group (P < 0.001) and between the tumor group and tumor control group (P < 0.001). However, p16 nuclear expression showed no significant difference between tumor control and normal control groups (P = 0.48). We also found a significant direct relationship between the intensity of p16 nuclear expression and the number of involved lymph nodes (P < 0.001). However, significant relationship was not found between p16 nuclear expression and age, tumor size, tumor grade, and carcinoma subtype (P > 0.05).
The key role of p16 as a regulator of the cell cycle results in a significant impact of its altered expression on pathological variables and the clinical course of a variety of human cancers.[151826] However, there is not much data on p16 expression in normal breast tissue and various kinds of breast lesions and the relationship between p16 expression in breast cancer and significant clinicopathological variables of this tumor. Feriancová et al. examined COX-2, p16, and Ki67 expression in ductal intraepithelial neoplasia (DIN), invasive breast cancer, benign breast lesions, and normal tissue adjacent to breast cancer. They found p16 overexpression in 37% of invasive breast carcinomas and 8% of normal tissue adjacent to carcinoma.[19] Golmohammadi et al. found P16 overexpression in 82% of breast cancers. No p16 overexpression was seen in normal breast samples. Overexpression of p16 had a significant association with higher tumor grade and tumor stage.[27] The study of Bazarov et al. on two malignant human breast cancer cell lines showed individual RB family proteins to be sufficient for p16-initiated senescence establishment. Although we found a significant direct relationship between the intensity of p16 nuclear expression and lymph node involvement in breast cancer, Dublin et al. did not find any relationship between p16 staining and histopathological parameters of invasive breast carcinoma. Geradts et al. also found no significant correlation between abnormal p16 expression in invasive breast cancer and several histopathological parameters of this tumor. Gorgoulis et al. found aberrant expression of p16 in 47% of breast carcinomas. However, they found no significant relationship between p16 expression and tumor size, lymph node metastasis, tumor grade, tumor stage, estrogen receptor (ER), and progesterone receptor (PR). Grupka et al. found breast cancers negative for both pRb and p16 to be associated with a better prognostic phenotype.[2829303132] Shin et al. found a significant correlation between p16 negativity and ER negativity, PR negativity, and higher Ki67 labeling index, all of which are linked to more aggressive breast cancer behavior.[16] In the study of Hui et al., p16 overexpression showed a significant association with high tumor grade, metastasis to axillary lymph nodes, ER negativity, and increased risk of relapse. Milde-Langosch et al. found a significant association between p16 overexpression and unfavorable prognostic indicators.[3334] According to the findings of Shan et al., p16 expression in luminal-A breast cancers is associated with progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma, and p16 expression is important for the development of triple-negative breast cancers.[35] In the study of Zhang et al., p16 expression inhibited breast cancer cell-induced angiogenesis and suppressed breast tumor metastasis in a spontaneous metastasis model in mice.[36] Radisky et al. found that p16 overexpression does not significantly stratify breast cancer risk in women with atypical ductal hyperplasia.[37] In the study of Naji-Haddadi et al., p16 positivity in breast cancer was not associated with tumor grade, tumor size, neural and vascular invasion, and lymph node metastasis.[38] Salih et al. reported an association between p16 expression in breast cancer and high histologic grade and lymph node metastasis.[39] According to the findings of Jovanovic et al., p16 protein has an important role in proliferation and malignant transformation, as well as in the progression from non-invasive breast lesions to invasive breast cancer.[40]
The different results of various studies may be attributed to different specificity and sensitivity of various antibodies, duration of fixation with its impact on the results of p16 immunohistochemical staining, and genetic differences among different populations. Despite these discrepancies, almost all studies including our study show a considerable frequency of p16 overexpression in breast cancer. Although the existence of a significant relationship between p16 overexpression in breast cancer and clinicopathological prognostic factors is not confirmed by all studies, at least some of them including our study confirm the presence of such a relationship between p16 overexpression and some clinicopathological prognostic factors. These findings suggest p16 as a potential biomarker for targeted therapy of breast cancer in future. Further studies are needed to examine this possibility.

CONCLUSION
This study suggests p16 overexpression as an important step in the malignant transformation of normal breast epithelial cells. Concerning the relationship between p16 overexpression in invasive breast carcinoma and clinicopathological prognostic factors, we only found a significant direct relationship between overexpression of this marker and metastatic involvement of axillary lymph nodes.

Financial support and sponsorship
Nil.

Conflicts of interest
The Deputy of Research of Isfahan University of Medical Sciences, Isfahan, Iran, has approved and financially supported this study.