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ORIGINAL ARTICLE
Year : 2022 | Volume
: 13
| Issue : 3 | Page : 213-224
Expression of poly(Adenosine Diphosphate-Ribose) polymerase protein in breast cancer
Akanksha1, Shashi Prakash Mishra1, Amrita Ghosh Kar2, JS Karthik1, Aviral Srivastava1, Rahul Khanna1, Ram Niwas Meena1
1 Department of General Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, UP, India
2 Department of Pathology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, UP, India
Correspondence Address:
Ram Niwas Meena
Department of General Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005, Uttar Pradesh
India
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/jmh.jmh_132_22
Abstract
Background: The use of poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors for breast cancer (BC) therapy is the subject of debate, and there is an urgent need to understand much the expression and prognostic role of the PARP1 protein. In this study, we have compared the expression of PARP between BC and benign breast disease (BBD) patients and also analyzed the association of PARP expression with clinicopathological parameters in BC. Methods: The study consists of 30 patients with newly diagnosed operable BC who were planned for surgery without neoadjuvant chemotherapy and 15 patients of BBD as a control between 2019 and 2021. Immunohistochemical analyses were performed prospectively on tissue samples. Anti-human PARP1 rabbit polyclonal antibody gives strong nuclear positivity. Internal control was the adipose tissue and the BBD acted as the external control. PARP1 expression was evaluated using the multiplicative quickscore method. Results: The mean age for BC patients was 51.30 ± 10.694 years (range: 25–75 years) while BBD was below 30 years. Overexpression of PARP was present in 25 (83.3%) and weak expression in 5 (16.7%) of BC patients compared to BBD, only 2 (13.3%) patients demonstrated an overexpression of PARP, and 13 (86.6%) patients showed weak expression which showed significant association (P < 0.001). In BC, nuclear PARP (nPARP) overexpression was seen in 22 (73.3%) patients and weak expression of nPARP in 8 (26.7%), whereas 5 (16.7%) patients showed cytoplasmic overexpression. On comparing expression of PARP with clinicopathological parameters, PARP overexpression was significantly associated with older population (age >50 years) (P = 0.002), postmenopausal women (P = 0.029), higher TNM stage (Stage II and III) (P = 0.014), higher histological grade (grade 2) (P = 0.043), and presence of lymphovascular invasion (P = 0.015). Enhanced PARP1 expression is closely correlated with positive estrogen receptor status (P = 0.001) and PR status (P = 0.001). Overall PARP and nPARP overexpression was significantly associated with ER- (P = 0.006 and P = 0.008) and PR-positive (P = 0.006 and P = 0.008) patients. The PARP and nPARP overexpression was significantly associated with nontriple-negative BC patients (P = 0.001 and P = 0.001). Conclusion: We have not come across any study in the literature to compare PARP expression in BC and BBD patients. On the basis of our observations, we concluded that PARP overexpression is a poor prognostic marker in BC.
Keywords: Benign breast disease, breast cancer, immunohistochemistry, poly(adenosine diphosphate-ribose) polymerase
Introduction 
Breast cancer (BC) is the second leading cause of cancer-related death in women and its incidence rates are further increasing.[1] There were approximately 2.3 million cases (11.7% of all cancers) and 0.6 million deaths (6.9% of all cancer-related deaths) in 2020 worldwide.[2] As per the WHO, in 2020, there were 2.3 million women diagnosed with BC and 685,000 deaths that occurred globally. By the end of 2020, there were 7.8 million women alive who were diagnosed with BC in the past 5 years, making it the world’s most prevalent cancer.
In India, approximately 25% of the female cancer cases are BC,[3] although the age-adjusted incidence rate of BC is lower (25.8 per 100,000) than the United Kingdom (95 per 100,000), mortality is at par (12.7 vs. 17.1 per 100,000) with the United Kingdom. According to GLOBOCAN 2012, India along with the United States and China collectively accounts for almost one-third of the global BC burden. Indian women having BC are found a decade younger in comparison to Western women suggesting that BC occurs at a younger premenopausal age in India and tends to be more aggressive. The main reason for this observed hike in mortality is due to lack of inadequate BC screening, diagnosis of disease at advanced stage, and unavailability of appropriate medical facilities.
The objective of the WHO Global Breast Cancer Initiative is to reduce global BC mortality by 2.5% per year, thereby averting 2.5 million BC deaths globally between 2020 and 2040. Reducing global BC mortality by 2.5% per year would avert 25% of BC deaths by 2030 and 40% by 2040 among women under 70 years of age. The three pillars toward achieving these objectives are health promotion for early detection, timely diagnosis, and comprehensive BC management.[4]
BC is a highly heterogeneous disease with variable histopathological and biological features and with different clinical outcomes and responses to therapy. A variety of clinical and pathological factors are routinely used to categorize BC patients in order to assess the prognosis and determine the appropriate therapy.[5] However, with the traditional diagnostic tools, patients with the same clinicopathological parameters can have markedly different clinical courses. Thus, the search for novel potential biomarkers to improve the clinical management of BC patients is an ongoing task.
Poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) plays important roles in DNA damage response pathways and is often overexpressed in various human tumors. PARP inhibitors have gained recent attention as promising therapeutic agents for the treatment of solid tumors including BC. The potential role of PARP inhibitors as a new anticancer agent is currently under investigation in several phase I to III studies in BC.[6],[7],[8],[9] However, the biological and clinical significance of PARP1 expression in BC and its role in DNA-damage response remain to be defined.
Immunohistochemical assessment of PARP1 reactivity in BC cells might be a potential biomarker able to provide insight into disease prognosis and response to therapy. An increased PARP1 expression has been demonstrated in different primary human tumors compared to normal tissue counterparts.[10],[11],[12] In BC patients, various authors have investigated the prognostic role of the PARP1 protein in relation to its different localization (nucleus and cytoplasm), demonstrating that PARP1 expression seems to be linked with a poor clinical outcome.[13],[14],[15],[16] In other studies, its value as a poor prognostic marker has not been revealed.[17],[18],[19],[20] Hence, it is important to clarify the importance of PARP1 expression in BC. Currently, the use of PARP inhibitors for BC therapy is the subject of debate, and there is an urgent need to understand much the expression and prognostic role of the PARP1 protein.
In this study, we have compared the expression of PARP between BC and benign breast disease (BBD) patients and also analyzed the association of PARP expression with clinicopathological parameters in BC.
Methods The cohort used in this study consists of 30 patients with newly diagnosed operable BC who were planned for surgery without neoadjuvant chemotherapy and 15 patients of BBD operated for fibroadenoma as a control between 2019 and 2021 in the Department of General Surgery, Institute of Medical Sciences, Banaras Hindu University. The ethical approval of the study was take from the Institute Ethical Committee. The protocols of study were in accordance to the ethical guidelines of Declaration of Helsinki, 1975. Proper written informed consent was taken from all the patients. The patients with a previous or concomitant history of beast cancer or other previously treated malignancies, metastatic diseases (Stage IV), pregnant or immunocompromized, were excluded from the study. All the patients underwent a thorough clinical history and examination, routine blood tests, X Ray Chest, Ultrasound abdomen and breast and a core needle biopsy from the breast lump. The plan of study is summarized in [Figure 1].
Biopsy sample/resection specimens were fixed in 10% buffered formalin and embedded in paraffin. The specimens collected are processed for detailed histological study. The hematoxylin- and eosin-stained slides are observed under light microscopy. The histological details supporting the prognostic parameters such as type of malignancy, differentiation (grade), lymphovascular and perineural invasion, lymph node status, and size of tumor are recorded. Each tumor is then graded according to the Nottingham Histology Score.
Immunohistochemical analyses were performed prospectively on tissue samples. Formalin-fixed, paraffin-embedded tissue sections were freshly prepared (2–4 mm) and subsequently incubated for 1 h at 60°C–62°C and then put in xylene for 15–20 min. Hydration is carried out at 100%, 90%, and 70% alcohol each for 1–2 min and washed. Antigen retrieval is done using citrate buffer at pH = 6.0. Slides are left for cooling at room temperature and washing is done. After endogenous peroxidase activity blocking for 10 min with 3% H2O2, the sections were incubated with anti-human PARP1 rabbit polyclonal antibody (VITRO Master Diagnóstica, Spain) by putting on the slide (overnight at 4°C). Super-enhancer is then put and kept for 30 min. Label (secondary antibody) is put on the slide and kept for 30 min. Diaminobenzidine is put and kept for 1–10 min until brown color develops and then put in distilled water to stop reaction. Slides are then counterstained with Harris hematoxylin for 10–15 s and differentiation is done with 1% acid-alcohol. Slides are then blotted, dried, and mounted with DPX and light microscopy performed.
Anti-human PARP1 rabbit polyclonal antibody gives strong nuclear positivity. Internal control was the adipose tissue and the BBD-like fibroadenoma and benign phyllodes acted as the external control.
All stained slides are scored in a blind manner by two independent investigators who had no previous knowledge of the clinicopathological data. PARP1 expression was evaluated using the multiplicative quickscore method (QS),[21] a system that accounts for both the intensity and the extent of cell staining. In brief, the proportion of positive cells was estimated and given a score on a scale from 1 to 6 (1 = 1%–4%, 2 = 5%–19%, 3 = 20%–39%, 4 = 40%–59%, 5 = 60%–79%, and 6 = 80%–100%). The average intensity of the positive-staining cells was given a score from 0 to 3 (0 = no staining, 1 = weak, 2 = intermediate, and 3 = strong staining). The QS was then calculated by multiplying the percentage score by the intensity score, to yield a minimum value of 0 and a maximum value of 18. Based on the QS, PARP1 expression was graded as low (0–9, subsequently mentioned as negative) or high (10–18, subsequently mentioned as positive).
Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS), Version 23.0. IBM Corp., NY, USA. Simple descriptive statistics were used (mean ± standard deviation) for quantitative variables, and frequency with percentage distribution for categorized variables. For categorical data, Chi-square test and Fisher’s exact test were used. For comparing two groups of mean, an independent Student’s t-test was used. For paired samples, paired t-test was applied for statistical analysis. The critical value of “P” indicating the probability of significant difference was taken as <0.05 for comparison. The critical value of “P” indicating the probability of significant difference was taken as <0.05 for comparison.
Results The mean age of BC patients was 51.30 ± 10.694 years (range: 25–75 years) and all patients of BBD included as a control were below 30 years. Majority (60%) of cases were above 50 years of age followed by age group between 31 and 49 years (33.3%) and <30 years (6.67%), respectively. In BC, 25 patients were female and 5 were male, while in BBD (controls), all were female. Majority of the patients belonged to locally advanced BC with 19 (63.3%) patients being in Stage IIA and IIB, 5 (16.7%) in stage IIIA/IIIB, and 6 (20%) patients in clinical Stage I as per TNM staging of AJCC 8th (2017). The most common histological type was invasive ductal carcinoma, not otherwise specified accounting for 27 (90%) cases, invasive lobular carcinoma was seen in 2 (6.67%), and invasive ductal carcinoma with neuroendocrine differentiation was present in 1 (3.3%) patient. According to the Modified Bloom-Richardson grading, the most predominant histological grade encountered was Grade II in 17 (56.7%) patients followed by Grade III in 7 (23.3%) and Grade I in 6 (20%), respectively. Lymphovascular invasion was noted in 20 (66.7%) specimens of Modified radical mastectomy (MRM) and 9 (30%) showed the presence of perineural invasion. Estrogen receptor (ER) and/or progesterone receptor (PR) positivity was present in 19 (63.3%) cases and overexpression of human epidermal growth factor receptor 2 (HER2)/neu was seen in 6 (20%) cases while 24 (80%) patients showed weak expression [Figure 2]. Nineteen (63.3%) patients were in luminal A and luminal B, 9 (30%) patients were basal cell like (BCL) (triple-negative phenotype), and only 2 (6.67%) patients were HER-2/neu enriched. The demographic and clinicopathological characteristics of patients are shown in [Table 1].
Figure 2: Histopathological images showing (a-d). H and E staining of invasive ductal carcinoma breast; Immunohistochemical images of nuclear staining showing ER positive; PR-positive nuclear staining; HER2/neu overexpression. HER2: Human epidermal growth factor receptor 2, ER: Estrogen receptor, PR: Progesterone receptorOverexpression of PARP was present in 25 (83.3%) patients and weak expression in 5 (16.7%) patients of BC compared to the control group (BBDs), only 2 (13.3%) patients demonstrated an overexpression of PARP, and 13 (86.6%) patients showed weak expression of PARP which showed a statistically significant association (P < 0.001) [Table 2]. In BC, nuclear PARP (nPARP) overexpression was seen in 22 (73.3%) cases and weak expression of nPARP in 8 (26.7%), whereas 5 (16.7%) patients showed cytoplasmic overexpression [Figure 3] and [Figure 4].
Figure 3: Overexpression of PARP in carcinoma breast patients. PARP: Poly(adenosine diphosphate-ribose) polymerase
Figure 4: Immunohistochemical images showing invasive ductal carcinoma breast with nPARP overexpression in 60%–70% cells (a) ×10 magnification; (b) ×40 magnification. nPARP: Nuclear PARP
Table 2: Comparison of poly(adenosine diphosphate-ribose) polymerase expression between breast cancer and benign breast disease patientsOn comparing expression of PARP with clinicopathological parameters, we found PARP overexpression was significantly associated with older population (age >50 years) (P = 0.002), postmenopausal women (P = 0.029), higher TNM stage (Stage II and III) (P = 0.014), higher histological grade (Grade 2) (P = 0.043), and presence of lymphovascular invasion (P = 0.015) in MRM specimen [Table 3]. Enhanced PARP1 expression is closely correlated with positive ER status (P = 0.001) and PR status (P = 0.001). No statistically significant correlation was identified between PARP1 expression and HER-2 status (P = 0.221) [Table 3].
Table 3: Expression of poly(adenosine diphosphate-ribose) polymerase with various clinicopathological parameters in carcinoma breastOn comparing overall PARP, nPARP, cytoplasmic PARP expression with ER, PR, and HER2/neu status in carcinoma breast patients, overall PARP and nPARP overexpression was significantly associated with in ER-positive patients (P = 0.006 and P = 0.008), but expression of cPARP was not significantly associated in ER-positive patients (P = 0.396) [Table 4]. Similarly, the overall PARP and nPARP overexpression was also significantly associated in PR-positive patients (P = 0.006 and P = 0.008), but expression of cPARP was not statistically associated in ER-positive patients (P = 0.396) [Table 4]. However, the overall PARP, nPARP and cPARP overexpression was not significantly associated with HER2/neu status in BC patients (P = 0.596, P = 0.251, and P = 0.221, respectively) [Table 4].
Table 4: Comparison of overall poly(adenosine diphosphate-ribose) polymerase, nuclear poly(adenosine diphosphate-ribose) polymerase, cytoplasmic poly(adenosine diphosphate-ribose) polymerase expression with estrogen receptor status, progesterone receptor, and human epidermal growth factor receptor 2/neu status in carcinoma breast patientsOn comparing overall PARP expression between triple-negative and nontriple-negative cases, all the nontriple-negative cases had PARP overexpression which showed a statistically significant association (P = 0.001). On comparing nPARP expression between triple-negative and nontriple-negative cases, 19 (90.5%) nontriple-negative cases had PARP overexpression which showed a statistically significant association (P = 0.001). Cytoplasmic PARP expression was not statistically associated with triple-negative breast cancer (TNBC) and non-TNBC cases [Table 5].
Table 5: Comparison of overall poly(adenosine diphosphate-ribose) polymerase, nuclear poly(adenosine diphosphate-ribose) polymerase, cytoplasmic poly(adenosine diphosphate-ribose) polymerase expression in triple-negative breast cancer, and nontriple-negative breast cancer patients Discussion BC is the most common malignant tumor and the second leading cause of cancer death in women worldwide.[22] As per the GLOBOCAN 2018 (under the International Agency for Research on Cancer) data, 162,468 women were newly detected with BC in India in the year 2018. This accounted for 27.7% of all newly detected cancers in women, carrying a mortality of 23.5% among all cancer-related deaths in women in India.[23] Its mortality is beginning to decrease due to early detection and improved therapies.[24]
BC is a highly heterogeneous disease with variable histopathological and biological features and with different clinical outcomes and responses to therapy. A variety of clinical and pathological factors are routinely used to categorize BC patients in order to assess the prognosis and determine the appropriate therapy.[25] These include patient age, axillary lymph node status, tumor size, histological features (especially histological grade and lymphovascular invasion), and hormone receptor and HER2 status. However, with the traditional diagnostic tools, patients with the same clinicopathological parameters can have markedly different clinical courses. Thus, the search for novel potential biomarkers to improve the clinical management of BC patients is an ongoing task.
Studies using molecular biology techniques confirmed that BC is a complex disease whose heterogeneity and clinical-therapeutic implications must be determined by new prognostic and predictive markers to personalize therapy to individual patients. Identification of BC subgroups with a different clinical course and a different response to systemic treatment is crucial for the improvement of therapy results and will help introduce new, molecularly targeted treatment options to the standard regimen. In line with the St. Gallen expert consensus,[26] the evaluation of ER, PR, HER-2 status, and proliferation index measured by Ki-67 immunoreactivity is key as regards prognostic value, in addition to TNM classification.
PARP1 was identified by Chambon et al. in 1963[27] as a protein whose enzymatic activity allows it to generate ADP-ribose polymers. Gene encoding PARP1, one of the housekeeping genes, sits on the long arm of chromosome 1 and spans 23 exons.[28] PARP1 protein comprises three main domains: (i) DNA binding domain, located at the N-terminal end of the protein; (ii) automodification domain; and (iii) catalytic domain, which spans the C-terminus of PARP1 molecule.[29],[30] PARP1 is the main member of the PARP family. PARP1 induces cell survival through DNA repair; however, during apoptosis, PARP1 is cleaved into two fragments by caspases resulting in its inactivation.[31] This caspase-mediated PARP1 inactivation suggests that blocking PARP1 activity is vital for the proper function of the apoptotic machinery by the ensuing DNA fragmentation.[32] Although overexpression of PARP1 is found in different primary human tumors compared to normal tissue counterparts,[10],[12] the biological and clinical significance of PARP1 protein expression in BC remains to be fully elucidated. The potential role of PARP inhibitors as a new anticancer agent is currently under investigation in several phase I to III studies in BC.[10],[12] In this study, we have assessed the expression of PARP in BC and BBD patients and also analyzed the association of PARP expression with clinicopathological parameters in BC.
In the present study, 30 patients of carcinoma breast and 15 of BBD as control were taken and studied for their demographic data, clinicopathological profile, and expression of PARP and its significance with the various clinicopathological parameters.
In our study, the mean age of BC patients was 51.30 ± 10.694 years (range: 25–75 years) and all patients of BBD included as a control were below 30 years of age. Majority (60%) of cases were above 50 years of age followed by age group between 31 and 49 years (33.3%) and less than 30 years (6.67%), respectively. Similarly, in an Indian study,[33] the mean age of presentation of BC in India was found to be <50 years, which is lower than that in the developed countries (median age of 52 years, Curigliano et al.[34]). This suggests that BC occurs at a younger age group in India as compared to their Western counterparts.[35] Our result was also consistent with the Geenen et al.[23] data wherein the incidence of BC in the younger age group (25–49 years) accounted for almost 37.7% of all cases. Peak in occurrence of BC was seen in the age group of 50–69 (which accounts for almost 46.5% of all cases).
In our study, out of 30 patients, 22 (83.33%) patients were female and 5 (16.7%) patients were male in the carcinoma breast group, and in the BBD group, all the patients were female. Thirteen of the 25 female (52%) BC patients were postmenopausal in our study. Most of these female patients were multiparous, and only 2 (8%) were nulliparous. Breastfeeding was done by 23 (92%) out of the 25 female patients. The duration of disease was <5 months in 24 (78.6%) patients, but 6 (21.4%) also presented with a disease duration of more than 5 months. The mean body mass index of our patients was 23.28 ± 3.10 kg/m2 (range: 17.2–29.9 kg/m2).
All patients in our study had presented with breast lump (100%), followed by complaints of pain in breast in 10 (33.3%), ulceration in 5 (6.67%), and nipple discharge in 3 (10%). This was similar to the study done by Koo et al.,[36] who found that breast lump was the most frequent (83%) complaint, followed by nonlump breast symptoms such as nipple abnormalities (7%) and breast pain (6%) and nonbreast symptoms like back pain (1%) and weight loss (0.3%). Chalasani[37] found that the most common symptoms in the patients of breast cancer were irregularly shaped lump in the breast or armpit, changes in the shape, or size of the breast or nipple, skin dimpling or tethering, or alterations in the appearance of the nipple, or a bloody fluid coming from the nipple and in advance stages bone pain, weight loss may be seen.
On examination, 17 (56.7%) patients presented with a lump size of <5 cm whereas 13 (43.3%) with a size of more than 5 cm in our study. It was also noted that 19 (63.3%) patients had lumps situated in upper-outer quadrant, followed by central location in 7 (23.3%) patients. This was similar to the study done by Sohn et al.,[38] who observed 58% of breast lumps originated from the upper-outer quadrant or axillary tail, whereas the remainder were found at the nipple complex 9%, upper-inner quadrant 14%, lower-inner quadrant 9%, and lower-outer quadrant 10%. Upper-outer quadrant lesions were associated with an independent contribution toward a survival benefit.
Most of the patients in our study belonged to locally advanced carcinoma breast, with 19 (63.3%) patients being in Stage IIA and IIB, 5 (16.7%) in Stage IIIA/IIIB, and 6 (20%) patients in clinical Stage I. Despite this, all patients underwent modified radical mastectomy due to operability of lump as seen by the lump-to-breast ratio. The most common histological type was invasive ductal carcinoma, not otherwise specified accounting for 27 (90%) cases, invasive lobular carcinoma was seen in 2 (6.67%), and invasive ductal carcinoma with neuroendocrine differentiation was present in 1 (3.3%) patient. Similar data were obtained from a study by Weigelt et al.,[21] who noticed invasive ductal carcinoma NOS in 78% and invasive lobular carcinoma in 11.1% with invasive ductal carcinoma with neuroendocrine differentiation to be extremely rare (0%).
In the present study, according to the Modified Bloom-Richardson grading, most predominant histological grade encountered was Grade II in 17 (56.7%) patients followed by Grade III in 7 (23.3%) and Grade I in 6 (20%), respectively. Lymphovascular invasion was seen in 20 (66.7%) specimens of MRM, and 9 (30%) showed the presence of perineural invasion. Hormone receptor status was evidenced as ER and/or PR positivity in 19 (63.3%) cases, and overexpression of HER2/neu was seen in 6 (20%) cases while 24 (80%) patients showed weak expression of HER2neu status.
TNBC represents a consistent subgroup of BC with heterogeneous clinical presentation, clinical behavior, histology, and response to therapy. The etiological profile of TNBC that is associated with high mortality and insufficient therapeutic choices is little understood. TNBC accounts for 10%–20% of all BCs[39],[40] worldwide and nearly 30% among Asian women population.[41] From India, Patil et al.[42] observed 19.9% to be TNBC in a cohort of 683 BC patients. Similarly, Ghosh et al.[43] reported TNBC status in 29.8% of patients in 2008.
Because studies have shown that BC molecular classes vary, numerous attempts to validate and convert these molecular classes into defined groups can be discovered in routine practice.[44] The majority of classifications employed a combination of immunohistochemistry markers, such as ER, PR, and HER2, with or without other markers, such as basal and proliferation markers. Luminal A (ER + and/or PR+, HER2−), luminal B (ER + and/or PR+, HER2+), BCL (TNBC (Triple Negative Breast Cancer which are ER/PR/HER2 negative)), and HER2-positive tumors (ER−, PR−, and HER2+) were the four primary types of BC.[45] In our study, 19 (63.3%) patients were in luminal A and luminal B, 9 (30%) patients were BCL (triple-negative phenotype), and only 2 (6.67%) patients were HER-2/neu enriched. Similarly, in a study done on Indian population by,[46] which observed that the prevalence for luminal A, luminal B, HER2-enriched, and TNBC subtypes of BC were 33%, 17%, 15%, and 30% respectively, for luminal A, luminal B, HER2-enriched, and TNBC subtypes of BC.
Overexpression of PARP has been noted in several cancers, including BC.[47],[48] PARP1 overexpression was an independent adverse prognostic factor, and it may play a role in the clinical behavior of BC. In our study, overexpression of PARP was present in 25 (83.3%) patients and weak expression in 5 (16.7%) patients of BC compared to the control group of BBDs, only 2 (13.3%) patients demonstrated an overexpression of PARP, and 13 (86.6%) patients showed weak expression of PARP which showed a statistically significant association (P < 0.001). To the best of our searched knowledge, there was no study in the literature to compare PARP expression in BC and BBD patients, but our finding showed that PARP was overexpressed in malignant tumors including BC.
In our study, out of 30 patients of BC, PARP was overexpressed in 25 (83.3%) patients and weak expression was present in 5 (16.7%) patients. In a study by Rojo et al.[49], PARP was not overexpressed in 68.78% (227/330) and overexpressed in 31.21% (103/330) patients. In another study conducted by Siraj et al.,[15] PARP high expression was found in 44.7% (451/1008) patients and low expression in 55.3% (557/1008) patients. Mazzotta et al.[16] assessed the expression of the PARP1 protein in a cohort of 114 BC patients. They found that positive PARP1 nuclear immunoreactivity was found in 59.65% (68/114) of all cases. In turn, 40.35% (46/114) of tumors showed negative or low PARP1 immunoreactivity. On further classifying the type of PARP overexpression, nPARP overexpression was seen in 22 (73.3%) cases and weak expression of nPARP in 8 (26.7%), whereas only 5 (16.7%) patients showed cytoplasmic overexpression, while 25 (83.3%) had a weak expression of cPARP in our study. Mazzotta et al.[16] found that the majority of BCs exhibit a nuclear expression of the PARP1 protein, and the latter is associated with cytoplasmic localization in a small percentage of tumors. It is known that PARP1 is mostly localized in the nucleus and accounts for approximately 75% of overall PARP enzymatic activity.
On comparing expression of PARP with clinicopathological parameters, we found PARP overexpression was significantly associated with older population (age >50 years) (P = 0.002), postmenopausal women (P = 0.029), higher TNM stage (Stage II and III) (P = 0.014), higher histological grade (grade 2) (P = 0.043), and presence of lymphovascular invasion (P = 0.015) in MRM specimen. Similarly, Donizy et al.[50] found that PARP was significantly associated with older age (P = 0.025), higher tumor grade (P = 0.003), and more advanced clinical stage (P = 0.013). Siraj et al.[15] found that PARP overexpression was associated with aggressive clinical parameters such as larger tumor size (P = 0.0136), stage IV tumors (P = 0.0006), and grade 3 tumors (P < 0.0001). In our study, PARP overexpression was not significantly associated with tumor size (P = 0.249), histological type (P = 0.717), invasive ductal carcinoma not otherwise specified, histological node positive (P = 0.176), and perineural invasion (P = 0.593) which is consistent with the observation of von Minckwitz et al.[51] and Rojo et al.[49] and Donizy et al.[50]
In the present study, statistical analysis showed that enhanced PARP1 expression is closely correlated with positive ER status (P = 0.001) and PR status (P = 0.001). No statistically significant correlation was identified between PARP1 expression and HER-2 status (P = 0.221). Consistently with those of researchers, our results showed a positive association between PARP1 and ER expressions.[52] Some authors have examined the underlying mechanism of interplay between PARP1 and ER. They identified PARP1 as an important regulator of ER-dependent gene transcription, as a result of ER PARylation.[53],[54] The researchers showed that PARP1 overexpression in nuclear localization is closely correlated with positive ER and PR status which corresponds to our findings. In a study conducted by Rojo et al.,[49] Mazzotta et al.,[16] and Donizy et al.,[50] PARP overexpression was not significantly associated with PR, but in our study, PARP overexpression was significantly associated with PR status.
No statistically significant correlation was identified between PARP1 expression and HER-2 status (P = 0.221) in our study. Similar results as regards the lack of correlation of PARP1 overexpression with HER-2 status were obtained by Rojo et al.,[49] von Minckwitz et al.,[51] and Donizy et al.,[50] but Mazzotta et al.[16] found that PARP overexpression was significantly associated with HER-2/neu status.
In our study, hormone receptor (HR) positive was seen in 19 (63.3%) patients of carcinoma breast, and overall PARP (P = 0.006) and nPARP (P = 0.008) overexpression was statistically significant, but expression of cytoplasmic PARP was not statistically significant in HR-positive patients.
Green et al. in 2015[55] studied nPARP1 expression in its two forms, cleaved and noncleaved, and found out that PARP1c expression was positively associated with premenopausal younger age patients and higher tumor grade with nuclear pleomorphism and mitosis and carried a poorer overall prognosis. On the other hand, PARP1c expression was positively associated with the expression of ER and nontriple-negative tumors.
Out of 30 cases of BC, triple-negative BC was seen in 9 (30%) patients and nontriple negative in 21 (70%) patients. On comparing overall PARP expression between triple-negative and nontriple-negative cases, all the nontriple-negative cases had PARP overexpression which showed a statistically significant association (P = 0.001). On comparing nPARP expression between triple-negative and nontriple-negative cases, 19 (90.5%) nontriple-negative cases had PARP overexpression which showed a statistically significant association (P = 0.001). Cytoplasmic PARP expression was not statistically associated with TNBC and non-TNBC cases. Our finding of positive PARP1 expression in several subtypes of BCs, also reported by other authors,[16],[50] supports that PARP1 inhibitors may have promising applications in a larger fraction of patients, not only in BRCA-mutated or BRCAness tumors.
We acknowledge that our study has some limitations such as the lack of BRCA mutation testing done in our study, along with small sample size and no follow-up done regarding overall survival and disease-free survival in the patient population which could have further emphasized upon the prognostic significance of PARP1 expression.
Conclusion The expression of PARP1 was compared with various clinic pathological characteristics of BC patients and a statistically significant association was found between the expression of PARP1 and older patients, with age of more than 50 years, and postmenopausal status. A significant association was also seen with higher TNM stage of disease, higher histological grade, and peritumoral vascular invasion which are each independent poor prognostic markers for BC. Moreover, a significant association between ER and PR (P = 0.001) status was observed. A statistically significant correlation was seen with non-TNBC cases, which suggests that PARP1 inhibitors may have promising applications in a larger fraction of patients, not only in BRCA-mutated or BRCAness tumors. Finally, we concluded that PARP overexpression is a poor prognostic marker in BC.
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Conflicts of interest
There are no conflicts of interest.
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