PROGNOSTIC VALUE OF THE EXPRESSION OF ENDOGENOUS HYPOXIA ASSOCIATED PROTEINS HYPOXIA INDUCIBLE FACTOR-1 ALPHA (HIF-1Α) AND CARBONIC ANHYDRASE ISOFORM 9 (CAIX) EXPRESSIONS IN BREAST CARCINOMA.

Background: Hypoxia has been found to be related to malignant initiation, progression, increasing the occurrence of metastasis and therapy resistance in many cancer types, which made a real need for discovering drugs that could antagonize the bad effect of hypoxia in cancer, decide which patients will have benefit from such anti-hypoxia therapy then to monitor response to therapy, especially in breast carcinoma. It is important to detect degree of hypoxia in each cancer that could be done by evaluation of the expression of hypoxia-associated protein in cancer biopsies e.g. hypoxia inducible factor-1 alpha (HIF-1α) and carbonic anhydrase IX (CAIX) and their detailed role in breast cancer is still uncertain and gives conflicting results. Aim of the Work: Was to evaluate HIF-1α and CAIX expressions in breast carcinoma, correlating their expressions with each other, with presence of lymph node & distant metastases, with recurrence free and overall survival rates of breast cancer patients. Methods: We evaluated HIF-1α & CAIX expressions in sections from 90 paraffin blocks of breast carcinoma using immunohistochemistry . We analyzed correlations between their levels of expressions, clinic-pathological and prognostic parameters of our patients. Results: HIF-1α and CAIX positive expression in breast carcinoma was related to advanced stage, presence of lymph node metastases, HER2 amplified and triple negative molecular subtypes (p<0.001), higher tumor grade (p= 0.001 & 0.02 respectively) and negative ER (p= 0.005 & 0.008 respectively) & PR (p= 0.009 & 0.027 respectively) hormonal receptors, The expression of both markers was significantly positively correlated with each other (p<0.001). HIF-1α and CAIX positive expression in breast carcinoma was associated with shortened recurrence free and overall survival rates (p<0.001). Conclusion: HIF-1α and CAIX are markers of poor prognosis of breast carcinoma patients.

Background: hypoxia has been found to be related to malignant initiation, progression, increasing the occurrence of metastasis and therapy resistance in many cancer types, which made a real need for discovering drugs that could antagonize the bad effect of hypoxia in cancer, decide which patients will have benefit from such anti-hypoxia therapy then to monitor response to therapy, especially in breast carcinoma. It is important to detect degree of hypoxia in each cancer that could be done by evaluation of the expression of hypoxia-associated proteins in cancer biopsies e.g. hypoxia inducible factor-1 alpha (HIF-1α) and carbonic anhydrase IX (CAIX) and their detailed role in breast cancer is still uncertain and gives conflicting results. Aim of the work: was to evaluate HIF-1α and CAIX expressions in breast carcinoma, correlating their expressions with each other, with presence of lymph node & distant metastases, with recurrence free and overall survival rates of female patients with breast cancer. Methods: we evaluated HIF-1α & CAIX expressions in sections from 90 paraffin blocks of breast carcinoma using immunohistochemistry. We analyzed correlations between their levels of expressions, clinicpathological and prognostic parameters of our patients. Results: HIF-1α and CAIX positive expression in breast carcinoma was related to advanced stage, presence of lymph node metastases, HER2 amplified and triple negative molecular subtypes (p<0.001), higher tumor grade (p= 0.001& 0.02 respectively) and negative ER (p= 0.005& 0.008 respectively) & PR (p= 0.009& 0.027 respectively) hormonal receptors, The expression of both markers was significantly positively correlated with each other (p<0.001). HIF-1α and CAIX positive expression in breast carcinoma was associated with shortened recurrence free and overall survival rates (p<0.001). Conclusion: HIF-1α and CAIX are markers of poor prognosis of breast carcinoma patients. 952 activity of endogenous peroxidase; we exposed the sections to heat for antigen retrieval in the autoclave, incubated them overnight with primary mouse monoclonal anti-HIF-1α (Calbiochem, Germany, diluted 1:300), and primary rabbit polyclonal anti-CAIX (Santa Cruz Bioscience, Santa Cruz, CA, USA, diluted 1:100) antibodies at 4°C. We used the chromogen diaminobenzidine substrate (DAB). Lastly we counterstained sections with hematoxylin. We included positive and negative controls of both markers in all cases. We considered sections from cervical squamous cell carcinoma that was positive for HIF-1α, and CAIX as positive control for both markers [Lee et al., 2008)]. And we have omitted the primary antibodies and replaced the by non-immune serum for the negative controls.
Evaluation of immunohistochemical expression of HIF-1α and CAIX:-We considered any dark stained nuclei and positive membranous & cytoplasmic stain in >1% of the tumor cells as positive for HIF-1α and CAIX respectively [Trastour et al., 2007].

Results:-
Ninety females' patients were included in our study with 49 (54.4%) patients were >55years. All detailed clinicopathological criteria are included in table (1). Tables 2 &3; fig 1 HIF-1α positive expression in breast carcinoma was significantly correlated with older age of the patients, higher grade and advanced stage of the tumor. HIF-1α positive expression was significantly correlated with aggressive molecular subtypes as HER2 amplified and triple negative subtypes, presence of lymph node metastases, high KI67 index (p<0.001 for all of them), presence of distant metastasis (p=0.041), negative ER (p= 0.005) & PR (p= 0.009), but it had no significant correlation with histopathological subtype of breast cancer. Tables 2 &3; fig 2 The positive expression of CAIX in breast carcinoma was significantly correlated with older age of the patients, advanced stage of the tumor, aggressive molecular type, presence of lymph node metastases, high KI67 index, aggressive molecular subtypes as HER2 amplified and triple negative subtypes, (p<0.001 for all of them), higher grade (p=0.02) negative ER (p= 0.008) & PR (p= 0.027) hormonal receptors, But it had no significant correlation with histopathological subtype of breast cancer or presence of distant metastasis.

CAIX expression, correlation to clinical and histopathological findings
The expression of HIF-1α and CAIX in breast carcinoma was significantly positively correlated with each other (p<0.001).  Our present results detected that when HIF-1α positively expressed in breast carcinoma that will be significantly related to worse clinic pathological findings like older age of the patients, higher grade and advanced stage of the tumor, aggressive molecular subtypes, presence of LN and distant metastasis, also we found that cases with positive HIF-1α expression had a higher rate of carcinoma recurrence, poor RFS and 3 year OS rates.
Our results were near results of former research that was done by Nalwoga et al., 2016 that evaluate the expression of HIF-1α in relation to markers of angiogenesis and other clinicopathological criteria in a cohort of breast cancer from Africa and they detected that positive HIF-1α expression was associated with increased tumor angiogenesis, high cancer cell proliferation rate that was evidenced by increased Ki-67 labeling index, high cancer grade that points to HIF-1α as a poor prognostic marker of breast carcinoma and a therapeutic target for breast cancer patients. We detected an association between HIF-1α expression and the presence of LN & distant metastasis in breast carcinoma; our results were near results of Liu, et al., 2015 who had proved that proved that HIF-1α is a regulator of cell hypoxia response and focused on HIF-1α role in increasing breast carcinoma metastasis, as they explored that HIF-1 had several roles in metastasis, e.g. increasing malignant cells invasion, up-regulating epithelial-mesenchymal transition (EMT), and formation of metastatic niche. Liu, et al., 2015 also discuss the values of therapeutic benefits of targeting the HIF-1α for management of breast cancer patients that is considered a recent therapeutic approach that could be used in combination with currently used therapies.
Wigerup et al., 2016 found that positive HIF-1α protein expression is present in malignant tumors of many organs and that is associated with poor prognosis of carcinoma of cervix, endometrium and ovary. They stated that HIFs had many roles in cancer cells growth, proliferation, differentiation, angiogenesis, cancer cell metabolism, local invasion, lymph nodes and distant metastasis. Subsequently, HIFs could be responsible for to chemo-and radiotherapy resistance, so they are associated with poor prognosis of cancer patients.
In addition HIF-1α could increase the expression of PD-L1 that is an immune checkpoint protein, which could be responsible for immune suppression (Noman et al., 2014). Solid malignant tumors, like carcinoma of the breast contain hypoxic areas due to presence of vascularization defects in these rapidly growing cancer cells. HIF-1α plays an essential role in cancer cells adaptation to hypoxia by increasing transcription of many genes that could regulate angiogenesis, proliferation, invasion, and metastasis (Semenza, 2012).
Another mechanism by which HIF-1α can act is by up-regulating epithelial-mesenchymal transition (EMT) process which is essential for tumor progression. EMT could be stimulated by hypoxia, by many mechanisms like HIF-1α pathways in several human malignancies. HIF-1α induce EMT by up-regulation of EMT transcription factors e.g.

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Twist, Snail, Slug and Zeb in many cancer types , In addition, hypoxia is an angiogenesis stimulating agent via production of many HIF-1 transcription factors, moreover during the EMT process HIF-1α stimulated angiogenesis by up-regulating VEGF transcription, and associated with microvessel growth which is an evidence of activated angiogenesis [Wigerup et al., 2016].
As we found that increased positive HIF-1α expression shows strong association with poor outcome and dismal survival rates of breast carcinoma patients, so that hypoxia is considered a hallmark of aggressive behavior of many solid tumors and responsible for metastases and therapy resistance, so it is considered a cancer attractive therapeutic targets like the recently discovered HIF-1α inhibitors Liu, et al., 2015.
HIF-1α inhibitors, like digoxin and acriflavine, had potential therapeutic roles in decreasing cancer growth, invasion, metastasis and vascularization in breast cancer (Wong et al., 2012), HIF-1α targeting is considered as a novel therapeutic modality for management of breast cancer patients and improving their prognosis which could be used in combination with currently used therapies.
Many researchers have studied CAIX expression in a plethora of human malignancies and stated that it was associated with poor patient's outcome, but its role in breast carcinoma patients still needs further clarifications [Thiry et al., 2006].
Here we proved that positive CAIX expression in breast carcinoma tissues was correlated related to worse clinic pathological findings like older age of the patients, higher grade and advanced stage of the tumor, aggressive molecular subtypes, presence of LN metastasis, also we found that cases with positive CAIX expression had a higher rate of carcinoma recurrence, poor RFS and 3 year OS rates.
Glaberman et al., 2016 also found nearly the same, that CAIX expression was related to aggressive pathological phenotype, chemotherapy resistance and poor prognosis of patients with breast cancer.
We  That association of CAIX positive expression with aggressive clinicopathological and prognostic parameters of breast cancer patients that proved by our results and results of previous studies, could support the theory that discovering selective CAIX inhibitors could be used to manage cancer patients and improving their prognosis, moreover some of such inhibitors are in the preclinical setting and still under evaluation [Ward, et al Both markers, mainly HIF-1α, is involved in the key step of the metastatic process e.g. EMT, malignant cell invasion, and metastatic niche formation.
As we demonstrated that breast carcinogenesis is stimulated by cells adaptation to hypoxia and acidosis, moreover the glycolytic, acid-resistant phenotype that has HIF-1α and CAIX positive expression is an aggressive phenotype.
Hence it will be better that tumor management strategies should aim at antagonizing the sequence of hypoxia, glycolysis and acidosis.
Moreover, identification of the metabolic phenotype of breast carcinoma will allow discovering to novel therapeutic modalities.
Also, the aggressive triple negative molecular subtype that is difficult to treat, as they are both chemo-resistant and hormonal non-responsive, and as we detected that such subtype showed positive expression of both HIF-1α and its downstream target CAIX, so targeting them both e.g. targeting HIF-1α with its inhibitors, gene therapies and CAIX inhibitors could be of particular importance in managing this aggressive cancer and improving patients prognosis (Supuran, 2008). The combination of HIF-1α & CAIX inhibitors with existing therapeutic modalities might be found to be useful clinically.
Clinical therapeutic trials are needed to determine if they could increase the survival of patients having breast cancer alone or in addition to currently used therapies.
Future studies are needed to discover more specific HIF-1α & CAIX inhibitors, to study their detailed mechanism of action, and to include them in clinical therapeutic trials of breast cancer patients.