Tumor LDH-A expression and serum LDH status are two metabolic predictors for triple negative breast cancer brain metastasis

There are limited therapeutic methods for triple negative breast cancer in the clinic, which is easy to progress into the brain to form metastatic lesions and evolve into the terminal stage. Because both the primary cancer and the brain metastasis have high glycolysis, we hypothesize that lactate dehydrogenase (LDH), which catalyzes the final step of glycolysis, may be a predictor, as well as a treatment target, for breast cancer brain metastasis. Therefore, the expression of LDH-A was detected on 119 triple negative breast cancer tissues with immunohistochemistry, and the serum LDH levels were also measured. Our results showed that the LDH-A expression inside the tumor was significantly higher than the matched normal tissues. Tumor LDH-A expression, serum LDH status, and the slope of serum LDH status were closely associated with triple negative breast cancer brain metastasis and brain metastasis free survival. This study indicates that tumor LDH and serum LDH status are two predictors for triple negative breast cancer brain metastasis.

Tumor LDH-A expression is higher than the matched normal tissues. We obtained the tissue samples from the 119 enrolled patients, which included both the breast cancer tissues and the matched normal tissues. The tissue samples were stained under the same condition (Fig. 1). The paired sample t-test was applied to determine the significant difference (p < 0.001) between the two groups. A great proportion of cases (up to 57%, 68/119) showed negative LDH-A expression in the matched normal tissue and positive expression in the cancer tissue, while only 6% (7/119) of the whole samples showed positivity in the matched normal tissues and negativity in the cancer tissues. The details of the methods are described in the section of "Materials and Methods". The associations of brain metastatic status with tumor LDH-A expression, pre-operational serum LDH level, and serum LDH status (including the status evaluated by Method A and Method B) were calculated by chi-square test. Tumor LDH-A expression and serum LDH status categorized by Method A were found to be significantly associated with brain metastasis (p = 0.023, p < 0.001, respectively), but not the baseline serum LDH levels nor the LDH status determined by Method B (Table 1).

Tumor LDH-A expression, as well as the serum LDH status determined by both Method A and
Method B, is associated with the brain metastasis free survival. Brain

Discussion
Brain metastasis is the worst complication of breast cancer due to the short survival of the patients and limited therapeutic regimens. High glycolysis activity is a prominent feature of brain metastasis accompanied by high expression levels of glycolysis-related proteins. Patients with brain metastasis consist of more TNBC cases as shown in previous reports 16,18,37 . Therefore, all the patients in this study were with the molecular phenotype of triple negative breast cancer and with the histopathology of infiltrating ductal carcinoma, had accepted modified radical mastectomy, and had no diabetes. LDH-A expression depends on the functions of the tissues and the increases in responses to tissue injury, necrosis, hypoxia, and so on. The tissues in this study had not been injured before modified radical mastectomy. The elevated serum LDH is not only prevalent in human malignancies, but also related to tissue injury, hemolysis, and hepatic failure. Peripheral blood samples with relative normal transaminase levels and without hemolysis were collected [38][39][40] .
As an important checkpoint enzyme catalyzing the final step of glycolysis, LDH-A upregulation not only facilitates the anaerobic glycolysis in tumor cells and reduces their dependency on oxygen, but also produces more lactic acid. The upregulated glycolysis is an evolution result of cancer cells for the adaptation to hypoxia. However, it also has significant negative effects on normal cells for the increased lactic acid production leads to the significantly decreased extracellular pH. Normal cells, exposed in this low extracellular pH microenvironment for a long time, will enter necrosis or apoptosis through caspase-3-dependent and p53-dependent mechanisms 41,42 . On the other hand, lactic acid accumulation induces the degradation of the extracellular matrix, destroys the adjacent normal cell populations, and promotes angiogenesis. Therefore, the lactic acid accumulation catalyzed by LDH-A helps cancer cells break down the barriers, which are comprised of normal cells and extracellular matrix and are protective mechanisms against cancer metastasis, to fulfill the first step of cancer cell migration.
The LDH-A expression inside the cancer is a reflection of metabolic rates, and a high metabolic rate is the basic requirement of tumor proliferation. We found the LDH-A expression was positively correlated to tumor sizes, which indicated that it might influence tumor proliferation. Meanwhile, Ki-67 showed a distinctive association with tumor LDH-A expression. The proportion of Ki67 positive cancer cells was significantly reduced in the LDH-A negative tumor samples. This observation was consistent with previous studies, which showed that the small number of Ki67-positive cells are related to the slow growth of LDH-A deficient tumors. This phenomenon was also observed in the LDH-A knockdown tumor tissue in an in vitro study 37 .
The distinctively high expression of LDH-A in the breast cancer tissues compared with the matched normal tissues might indicate that the expression of LDH-A is a indicator of the malignancy degree. Another immunohistochemical study revealed that LDH-A is primarily expressed in cancer cells, whereas normal and carcinomatous tissues have similar levels of LDH-B, which is a member of LDH family 43 . LDH-B predominates in the tissues with an aerobic metabolism like the heart, while LDH-A is mainly present in the tissues with considerable anaerobic metabolism, such as the skeletal muscle, liver, and tumor. Furthermore, in a small patient cohort, the LDH-B levels were consistently low compared to the LDH-A levels 44 . These pieces of evidence enable LDH-A to be used as a biomarker for many malignancies [45][46][47] .
Tumor LDH-A expression, as a predictive biomarker, is the most straightforward. However, it will take a long time from the determination of tumor LDH-A expression to the development of brain metastasis. Serum LDH status can monitor the disease progression at the real time. Taken together, both LDH determination methods have their advantages and disadvantages. LDH-A expression inside the cancer tissue is not consistent with the baseline serum LDH levels in this study, which may indicate tumor LDH-A expression and serum LDH levels are two separated predictors. This result coincides with the view of Koukourakis et al. that the serum LDH-A levels were not correlated with cancer tissue levels in 71% of LDH-A positive cases 48 .
Serum LDH, which monitors the disease progression in the whole period, also showed significant associations with breast cancer brain metastasis. Serum LDH status categorized by Method A shows positive results, while the baseline serum LDH level has not. This result might be due to the fluctuation of serum LDH levels compared with the tumor LDH-A. Along with the enlarging tumor volume, glycolysis enhances, and the consequent increase in LDH expression leads to the elevation of serum LDH levels. On the other hand, the LDH released from the necrotic cancer tissue elevates the serum LDH level when the tumor volume exceeds the capacity of blood supply. Therefore, the tumor LDH-A levels and serum LDH level are two independent predictors for BCBM.
Measuring serum LDH levels is a safe and simple detection method compared with using biopsy. We show two methods of evaluating serum LDH status: Method A takes all serum LDH measurements during the disease progression, and Method B quantifies the serum LDH status at specified detection time points. Method A is better than Method B in predicting the occurrence of brain metastasis since the serum LDH levels evaluated by Method A shows relevance with brain metastasis but not Method B. The combination of tumor LDH-A expression and serum LDH levels evaluated by Method B shows a better prediction for BMFS in the LDH-A positive group, which suggest that Method B should be used when tumor LDH-A expression is positive. In the analyses of the relationship between tumor LDH-A expression or serum LDH levels with brain metastasis, both of them show a distinct association, which tells us both the indices could be predictors of breast cancer brain metastasis.
In summary, our result supports that LDH-A should be inhibited for BCBM prevention and treatment. The distinctively high expression of LDH-A in the breast cancer tissues is a indicator of the malignancy degree. As tumor LDH-A expression and serum LDH levels are two separated predictors, Method A is better than Method B in predicting the occurrence of brain metastasis, but the combination of tumor LDH-A expression and Method B shows a better prediction for BMFS in the LDH-A positive patients. We proved that tumor LDH-A expression, as well as serum LDH status, is associated with brain metastasis status.

Materials and Methods
Patients and tissue samples. 119 formalin-fixed paraffin-embedded breast cancer sections were obtained from The Third Affiliated Hospital of Harbin Medical University. These triple-negative patients were diagnosed between March 2005 and March 2015, who received standard treatment including chemotherapy and/or radiotherapy and had complete medical records. Furthermore, the patients were firstly diagnosed as triple-negative infiltrating ductal breast cancer without any severe systemic diseases or combined tumors and did not receive any treatments before the surgical operation. This study was approved by the Ethics Committee of the Third Affiliated Hospital of Harbin Medical University. Informed consent was obtained from all patients. All procedures were performed in accordance with the rules and guidelines of the Tumor Research Institute of Heilongjiang, Harbin, China. All participants in this study had signed informed consents.

Immunohistochemistry (IHC).
Formalin-fixed paraffin-embedded breast cancer sections were cut into four micrometer-thick sections and mounted on a slide for immunohistochemical staining. They were dewaxed, incubated in saline sodium citrate (pH = 7.0) for 1 min in pressure heating environment for antigen retrieval, then soaked in 3% H 2 O 2 solution for 30 min. Mouse monoclonal LDHA antibody (1:200, OriGene, USA) were applied at 4 °C overnight in humid chambers, followed by the secondary antibody at room temperature for 25 min. Then the color was developed by DAB. The stained specimens were reviewed by two pathologists independently. At least five visual fields were observed for each section under high power lens (×400) to calculate the percentage of positive cells (from an undetectable level (0%) to a homogeneous staining (100%)), and the intensity of staining was scored (1, weak staining; 2, moderate staining; and 3, strong staining). The scores were further calculated by multiplying the percentage of positive cells by the intensity (ranged from 0 to 300). The final score ≤100 was considered as the negative expression, and the score >100 was considered as the positive expression 49 . Serum LDH measurement. Blood samples from a peripheral vein puncture were collected before the operation and at each visit during the chemotherapy and follow-up period until brain metastasis development or the last follow-up. Hitachi Modular 7600 Chemistry Analyzer measures the LDH level of blood sample by spectrophotometry. LDH catalyzes lactates to pyruvates. During the reaction, NAD+ is reduced to NADH. NADH has an absorption peak at 340 nm wavelength, so the reaction increases the absorbance at 340 nm. The concentration of LDH can be determined according to the changes in absorbance at 340 nm. In the experiment, we Scientific REPORTS | 7: 6069 | DOI:10.1038/s41598-017-06378-7 first collected the blood samples from a peripheral vein puncture into vacutainer tubes (BD Vacutainer ® SST TM , 367983). Then, the blood samples were centrifuged at 3000 r/min for 5 min (Hitachi CT15RE Centrifuge) and put into the Hitachi Modular 7600 Chemistry Analyzer. The analyzer measured the LDH levels automatically. Based on the serum LDH levels before the treatment, a value of >246 U/L was considered as elevated level.
The evaluation of serum LDH status. Serum LDH levels were measured at every visit after the modified radical mastectomy until the development of brain metastasis or the last follow-up. The alteration status of serum LDH was evaluated in two ways. In the first method (Method A), the patients was classified into four subgroups according to their serum LDH levels: persistently normal, improved (the patient's LDH levels decreased from an elevated level to the normal level), deteriorated (the patient's LDH levels increased from the normal level to an elevated level), and persistently elevated. The baseline was the LDH level measured before the operation, and the LDH level was termed "elevated" at the terminal point if more than half of the measurements were higher than the baseline during the chemotherapy and follow-up.
In the second method (Method B), the serum LDH levels were measured at the surgery, before the first and fourth cycles of chemotherapy, and at the first follow-up after the chemotherapy. These time points corresponded to approximately at the surgery one month, three months, and seven months after the surgery, respectively. The values were used to calculate the slope of serum LDH changing curve by the least square method. Receiver operating characteristics (ROC) curves and the area under the curve (AUC) were used to determine the optimal cutoff points for the Method B. ROC curves, which help to choose the cut-points associated with optimal sensitivity and specificity, are commonly used in medical research to evaluate screening tests and identify thresholds to facilitate the decision making about patients 50 . Regarding the Method B in the TNBC patients, 5.0 U/L/month was identified as the optimal cutoff point for distinguishing the patients with a good prognosis from the patients with a poor prognosis (P = 0.048, AUC = 0.549), and the sensitivity and specificity were 44.4% and 68.1% (Fig. 3). All the patients were divided into two groups: the LDH high group (≥5.0 U/L/month) or the LDH low group (<5.0 U/L/ month). In addition, formalin-fixed paraffin-embedded breast cancer tissues were obtained from the modified radical mastectomy before any systemic treatments.
Statistical methods. All the data were analyzed with the statistical package SPSS (version 20.0 for Windows, Chicago, IL) software, and Pearson's chi-square tests were applied to study the association between LDH-A/LDH and other clinicopathological features of the enrolled breast cancer patients. The least square method was used to calculate the slope of serum LDH status and ROC to give the best cut-off of Method B. The LDH-A expression between the cancer tissues and the matched normal tissues was analyzed by paired samples t-test. The Kappa test and McNemar test were used to examine the consistency of tumor LDH-A expression levels and the baseline serum LDH levels. Survival curves were generated with the Kaplan-Meier method, and Cox's proportional hazards regression model was used to evaluate the hazards. Differences with p < 0.05 were considered statistically significant.
Data Availability Statement. All data generated or analysed during this study are included in this published article.