الفهرس 
EpidemiologOnly 14 pages are availabe for public view
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Abstract
Breast cancer (BC) represents the commonest malignancy among females in Egypt (38.8%) (Ibrahim et al., 2014) and around the world (Wilkinson and Gathani, 2022), with 2,088,849 (11.6%) new cases in 2018 (Ferlay et al., 2018). Series of recent research studies suggested the link between vitamin D deficiency and various adverse health outcomes, namely cardiovascular and cancer-related morbidity and mortality (Amrein et al., 2020). Nevertheless, evidence for a direct relationship between plasma 25(OH)D and breast cancer incidence and/or molecular subtype presentation remains conflicting. Vitamin D is thought to play a significant role in controlling normal breast cell growth and that it has the capacity to stop the growth of cancer cells in this location (Narvaez et al., 2014). This protective effect is believed to be largely supported by the chemo-preventive actions of 1,25-hydroxyvitamin D (1,25-(OH)2D, or calcitriol), the bioactive form of vitamin D, a well-known calcium regulator. Experimental, pre-clinical and ecological studies have shown that 1,25-(OH)2D induces differentiation and apoptosis and inhibits cell proliferation and angiogenesis in normal and malignant breast cells (Welsh, 2012, De La Puente-Yagüe et al., 2018). Similar associations have been reported in other observational studies (Huss et al., 2019, Fadavi et al., 2021). A recent meta-analysis showed that circulating levels of 1,25OH2D inversely correlate with the risk of breast cancer (Estébanez et al., 2018). The main criticism to most of the studies was that they weren`t powered to detect a statistically significant difference between the vitamin D deficient and sufficient patient groups. In our study, we evaluated the relationship and potential impact of vitamin D levels on the clinical outcomes of the newly diagnosed Egyptian breast cancer patients presenting to our department. We aimed to investigate the correlation between serum vitamin D levels and the clinicopathological features as well as the clinical outcomes of our patients mainly because numerous trials have been investigating the association between circulating vitamin D levels and breast cancer risk. Still, only a few have been designed to assess a possible relation of vitamin D with the prognostic features in breast cancer or with regard to the recent different molecular subtypes (Habib et al., 2014, De Sousa Almeida-Filho et al., 2017, Yao et al., 2017). Vitamin D deficiency is a common medical condition worldwide (Wahl et al., 2012, George et al., 2014, Li et al., 2014). Previous studies conducted in 18 countries located at different latitudes have shown that this is a phenomenon that also occurs in sunny regions (Amrein et al., 2020). Concordant with previous studies (Goodwin et al., 2009, Rejnmark et al., 2009, Vrieling et al., 2011), which reported 50–74% vitamin D deficiency in newly diagnosed breast cancer patients, our study demonstrated that 56.5% of patients with breast cancer had an insufficient level of vitamin D. When stratified by clinicopathological features in our study and compared to the patients with normal level of vitamin D, those with an insufficient levels of vitamin D at diagnosis had a greater risk of high grade (G3) disease (88.6%), higher rate of N2 and N3 lymph node status (86.2% and 75.5%, respectively), and T3 and T4 tumor size of (73.5% and 100%, respectively) when compared to patients with sufficient vitamin D levels. This is in accordance with literature data showing that locally advanced breast cancer patients have more severe vitamin D deficiency than those with early-stage disease (Hatse et al., 2012, Yao et al., 2017). Benedito de Sousa Almeida-Filho et al from Brazil conducted a cross-sectional analysis of vitamin D levels and clinicopathological features to evaluate the association between pretreatment vitamin D (VD) deficiency with breast cancer prognostic features in Brazilian postmenopausal women. Patients with insufficient and deficient 25(OH)D levels had a higher proportion of breast cancer of high grade and locally advanced and metastatic disease, more positive lymph node, a lower proportion of ER, PR positives tumors and higher Ki-67(p < 0.05) (De Sousa Almeida-Filho et al., 2017). Hatse et al. (2012) from Belgium, studied the correlation between serum 25(OH)D levels and tumor features and clinical disease outcome in 1800 early breast cancer patients. Lower 25(OH)D serum levels significantly correlated with larger tumor size at diagnosis but not with lymph node invasion, receptor status, or tumor grade. High serum 25(OH)D (> 30 ng/mL) at diagnosis significantly correlated with improved overall survival and disease-specific survival. However, when considering menopausal status, serum 25(OH)D had a strong impact on breast cancer-specific outcome in postmenopausal patients, whereas no association could be demonstrated in premenopausal patients (Hatse et al., 2012). In view of the increasing number of cancer survivors and the high prevalence of vitamin D deficiency among patients with cancer, an evaluation of the role of circulating 25-OHD in prognosis among these patients is crucial. It is suggested that lower serum vitamin D levels might have some contribution in the progression from early-stage to advanced disease due to the alteration of gene transcription, supporting the hypothesis that vitamin D deficiency has a role in the pathogenesis and progression of breast cancer (Holick, 2006). Vitamin D appears to exert antiproliferative effect on breast cancer, mediated by the regulation of the expression of molecules that control the progression of the cell cycle (Lopes et al., 2010, Narvaez et al., 2014). Malignant cells have decreased intracellular levels of 1α-hydroxylase (the activating enzyme encoded by CYP27B1) compared to normal cells, which increases intracellular vitamin D production (Thanasitthichai et al., 2015). Furthermore, some studies have demonstrated a reduction in vitamin receptor (VDR) expression in breast cancer cells compared to normal breast cells, by VDR gene polymorphisms (Costa et al., 2009, Engel et al., 2012). During malignant transformation of the breast, tumor cells lose the ability to synthesize the active form of vitamin D and to respond to the effects of vitamin D mediated by VDR [41]. In breast cancer, the expression of VDR varies from 58% to 80%, with reports of patients with VDR-positive tumors showing a higher disease-free survival rate [7]. In our study, 125 patients had vitamin D levels less than 30ng/L. Low Vit-D level was significantly more prevalent among triple negative breast cancer (TNBC) patients (7.6% vs 2.7% in deficient vs sufficient vitamin D level patients) compared to ER positive patients (32.13 vs 38.9%, respectively). Considering HER2- receptor status, 16.74% were deficient vs 1.8% with sufficient levels of vitamin D. And the difference among these subgroups was significant (p <0.001) In regard to prognostic biomarkers and their relationship with vitamin D level, similar results have been observed (Robien et al., 2007, Shirazi et al., 2016). Estrogen receptor (ER) and progesterone receptor (PR) have long been accepted as playing a central role in the pathobiology and treatment of breast cancer by predicting an indolent and slowly growing tumor with longer time to disease recurrence versus features of more aggressive nature (Prat et al., 2013, Widodo et al., 2017, Gao and Swain, 2018). Robien et al. (2007) suggested a strong relation between postmenopausal women with low vitamin D intake and higher risk of negative estrogen and progesterone receptor breast cancers (Robien et al., 2007). Shirazi et al. (2016) investigated the potential association between pre-diagnostic serum 25(OH)D levels and the risk of different subtypes of breast cancer. The study was based on The Malmo Diet and Cancer Study recruiting 17,035 women from 1991 to 1996. A total of 764 incident breast cancers with matched controls were analyzed for 25(OH) D in samples collected at baseline, before diagnosis. As compared to the first tertile of 25OHD3, the second tertile had a significantly lower risk of ER and PR-negative tumors and with a high expression of Ki-67. However, the third tertile had a similar risk as the first one (Shirazi et al., 2016). The relationship of vitamin D with breast cancer risk may be subtype-specific, with emerging evidence of stronger effects of vitamin D in more aggressive breast cancers It is appreciated that triple negative breast cancer, which lacks expression of ER and PR, does not have amplification of HER2 and expresses a high proliferation rate and is well known for its aggressive nature and poor prognostic outcomes (Curigliano et al., 2017). In our study, the majority of the triple negative and HER-2 positive patients were deficient in vitamin D (16.7% versus 1.81% and 7.69% versus 2.7% respectively). In the current literature, some studies have shown that while vitamin D deficiency is common in all breast cancer patient populations, it is particularly prevalent in those with these triple negative tumors (Laporta and Welsh, 2014). Park et al. (2015) in a large case–control study, compared serum 25(OH)D between breast cancer patients and general population. The adjusted odds ratio (OR) for breast cancer comparing women with deficient level of serum 25(OH)D to women with sufficient level of serum 25(OH)D was 1.27 (1.15–1.39). The association remained significant in both positive and negative hormone receptor (HR) status of the tumor. Noteworthy, this association was more pronounced in HR-negative breast cancer, particularly with triple-negative breast cancer patients (1.45, 95% CI1.15–1.82) (Yao et al., 2017). Since studies have demonstrated a high prevalence of vitamin D deficiency in women with basal-like breast cancer, correction of vitamin D deficiency in these women represents a reasonable, but yet untested, strategy to delay recurrence and extend survival (Ismail et al., 2018). As a strength of our study, the serum vitamin D was measured immediately after diagnosis of breast cancer and prior to any proposed treatment. In fact, some studies that have found an association between reduced values of 25(OH)D and a higher incidence of breast cancer performed the measurements of 25(OH)D during or after treatment, with a long interval from the diagnosis (Lowe et al., 2005, Abbas et al., 2008), which could have potentially been affected by the cancer treatments given. For instance, women with a lower physical activity rate have lower values of 25(OH)D, and it is well established that physical activity is consistently lower for years following a breast cancer diagnosis (Chlebowski, 2011). Vrieling et al. (2011) in a prospective cohort study, assessed the effect of post-diagnosis serum 25(OH)D on overall survival and distant disease-free survival in 1295 incident postmenopausal breast cancer patients (50–74 years), with a mean follow-up of 5.8 years. The association with overall survival was found to be significant only for 25(OH)D levels of blood samples collected before initiating chemotherapy, but not for those collected after chemotherapy had been administered (Vrieling et al., 2011). A meta-analysis, which correlated serum 25(OH)D with breast cancer risk, confirmed an inverse correlation in case-control studies that collected 25(OH)D immediately after breast cancer diagnosis, but not in prospective studies when 25(OH)D measurement was performed years before diagnosis (Yin et al., 2009). As breast cancer is a disease that develops over several years, a major assumption for a causal relationship between 25OHD levels and breast cancer is that 25(OH)D levels, in the individual subject, are at a steady level for a longer period of time. Thus, a limitation of our study is that only a single measurement of vitamin D at diagnosis was used, which might not necessarily represent vitamin D levels at the time of cancer initiation. However, in a cohort study, the correlation coefficient for measurement of 25(OH)D concentrations in serum samples collected in 1994 and 2008 ranged from 0.42 to 0.52 De Sousa Almeida-Filho et al. (2017) suggesting a reasonable stability of endogenous vitamin D status. However, more studies are needed to explore whether a single measurement of serum 25OHD level is a sensitive indicator of an individual long-term vitamin D status. In a small study by Ismail et al. (2018) from Egypt, that included 50 women with primary invasive, non-metastatic BC, patients were followed up for a median of 30 months. In this study, vitamin D deficiency was linked with worse overall survival (OS) and disease-free survival (DFS) (p = 0.026, and p = 0.004, respectively). On multivariate analysis, DFS was independently affected by vitamin D deficiency with an HR of 2.8 (95%CI: 1.6-7.0, p = 0.022) and advanced stage, i.e., stage II had worse survival compared to stage I with an HR of 4.8 (95%CI:1.1-21.7, p = 0.042) (Ismail et al., 2018). In another case-cohort study by Yao et al. (2017) similar results were also found; Among premenopausal women, the association with OS was stronger, and there were also associations with breast cancer–specific survival and invasive disease–free survival (OS: HR, 0.45; 95% CI, 0.21-0.96; breast cancer–specific survival: HR, 0.37; 95% CI, 0.15-0.93; invasive disease–free survival: HR, 0.58; 95% CI, 0.34-1.01; all after full adjustment). These results are concordant to the results from our study were DFS and OS were significantly worse in patients with vitamin D levels lower than 30 ng/l. A meta-analysis of 8 studies that investigated the association of circulating 25(OH)D concentrations with recurrence and mortality in breast cancer patients concluded that an increased serum level of vitamin D is associated with decreased risk of breast cancer and that vitamin D plays a significant role in protection of breast cancer (Obaidi et al., 2015). This association was further confirmed in another meta-analysis involving five studies with 4,413 breast cancer patients. The authors found that pooled hazard ratios for comparing highest with lowest vitamin D status categories were 0.62 (95% CI: 0.49-0.78) and 0.65 0.58 (0.38-0.84) for overall and disease-specific mortality, respectively (Maalmi et al., 2014). Another meta-analysis of 5 studies reported substantially lower mortality rates in individuals with higher serum concentrations of 25(OH)D (Yao and Ambrosone, 2013). In the AZURE-trial; a large, phase 3, international, multicenter, randomized, controlled clinical trial,, that included 3,360 women with stage II/III breast cancer where the role of adjuvant bisphosphonates in improving breast cancer outcomes in this group of patients was investigated, a subset analysis was conducted by Professor R Coleman from the united kingdom (UK), where two other markers of bone turnover tested, N-terminal propeptide type I procollagen (P1NP) and beta C-terminal telopeptide type I collagen (βCTX) where measured but failed to significantly differentiate breast cancer patients who may be at high risk for distant relapse. However, interestingly, the researchers found that 90 percent of study patients had insufficient vitamin D levels at diagnosis, an insufficient level was 30 ng/ml or less, and that patients who did have sufficient baseline vitamin D levels had a much better prognosis in terms of bone recurrence, with a hazard ratio of 0.11. Additionally, there was a trend toward better prognosis in time to distant relapse in this group with HR of 0.56. They presented their finding in the SAN-ANTONIO meeting in 2013 (Coleman et al., 2014). Further analysis of the clinical outcomes of our cohort of patients; there was a significantly higher incidence of the number of local recurrences, bone metastases, and liver metastases among the low vitamin D level group (p:<0.001). Interestingly; there was no significant difference between the two groups in terms of the development of lung or brain metastases. The association between vitamin D deficiency and bone metastases was suggested in a unique study of murine models by Ooi et al. (2010) were Vitamin D deficiency was found to enhance both the growth of tumors and the tumor-induced osteosclerotic changes in the tibiae of mice following intratibial implantation of MCF-7 cells. Enhancement of tumor growth appears dependent on increased bone resorption rather than increased bone formation induced by these tumors (Ooi et al., 2010). Another interesting study by Khadija El Abdaimi, reported the efficacy of EB 1089, a low calcaemic analogue of vitamin D, on the development of osteolytic bone metastases after intracardiac injection of the human breast cancer cell line MDA-MB-231 in nude mice where mice treated with EB1089 displayed a marked increase in survival and developed fewer bone lesions and less hind limb paralysis over time as compared with untreated animals (El Abdaimi et al., 2000). Up to our knowledge; no study, as yet, looked into the correlation of vitamin D deficiency and the incidence of local recurrence, nor visceral metastases. Vitamin D deficiency is prevalent among Egyptian non-metastatic breast cancer patients. Low serum Vitamin D in breast cancer patients negatively correlates with tumor size, lymph node positivity and stage at diagnosis. It is linked to HER-2 positive and TNBC, and is significantly associated with worse DFS and OS Our findings provide strong evidence that vitamin D can be used as a prognostic factor in patients with breast cancer, and clinicians should be screening their newly diagnosed breast cancer patients for their baseline vitamin D, among other prognostic and predictive markers. Although women with breast cancer will probably benefit in conditions of overall health from having high vitamin D levels, further investigation in more extensive in vitro and in vivo research is needed to investigate the impact of vitamin D levels correction on breast cancer outcomes.