Diagnostic and prognostic value of the cancer-testis antigen lactate dehydrogenase C4 in breast cancer

Journal Pre-proofs Diagnostic and prognostic value of the cancer-testis antigen lactate dehydrogenase C4 in breast cancer Zhaolei Cui, Yansong Chen, Minhua Hu, Yingfeng Lin, Shuyu Zhang, Lingying Kong, Yan Chen PII: DOI: Reference:

S0009-8981(19)32154-0 https://doi.org/10.1016/j.cca.2019.11.032 CCA 15942

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Clinica Chimica Acta

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6 August 2019 5 November 2019 23 November 2019

Please cite this article as: Z. Cui, Y. Chen, M. Hu, Y. Lin, S. Zhang, L. Kong, Y. Chen, Diagnostic and prognostic value of the cancer-testis antigen lactate dehydrogenase C4 in breast cancer, Clinica Chimica Acta (2019), doi: https://doi.org/10.1016/j.cca.2019.11.032

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Diagnostic and prognostic value of the cancer-testis antigen lactate dehydrogenase C4 in breast cancer

Zhaolei Cuia, Yansong Chena, Minhua Hua, Yingfeng Lina, Shuyu Zhanga, Lingying Kongb, Yan Chena a

Laboratory of Biochemistry and Molecular Biology Research, Fujian Provincial Key

Laboratory of Tumor Biotherapy, Department of Clinical Laboratory, Fujian Cancer Hospital & Fujian Medical University Cancer Hospital, Fuzhou, Fujian, P. R. China; b

Department of Pathology, Fujian University of Traditional Chinese Medicine

Affiliated People’s Hospital, Fuzhou, Fujian, P.R. China

Address correspondence to: Dr. Yan Chen, Department of Clinical Laboratory, Fujian Provincial Key Laboratory of Tumor Biotherapy, Fujian Cancer Hospital, Fujian Medical University Cancer Hospital, No. 420 Fuma Road, Jin’an District, Fuzhou 350014, Fujian Province, China. Tel: +86-59162752638, Fax: +86-59162752638, E-mail: [email protected]; Dr. Lingying Kong, Department of Pathology, Affiliated People’s Hospital of Fujian University of Traditional Chinese Medicine, 602 Middle Road, Fuzhou 350004, Fujian, China. Tel: +86-59183258135; Fax: +86-59183258135; E-mail: [email protected]. Abbreviations: AUC, area under the curve; BC, breast cancer; ER, estrogen receptor; LDHC, lactate dehydrogenase C; LDH-C4, lactate dehydrogenase C4.

Abstract Background: Lactate dehydrogenase C4 (LDH-C4) as a cancer/testis antigen (CTA) is abnormally expressed in some malignant tumors. However, the expression and clinical significance of LDH-C4 in breast cancer (BC) has not been characterized. Methods: We determined LDHC mRNA expression in serum and serum-derived exosomes of BC patients by quantitative RT-PCR. We also evaluated the protein expression of LDH-C4 in BC tissues using high-throughput tissue microarray analysis and immunohistochemistry. Results: Our results showed high mRNA expression level of LDHC in serum and serum-derived exosomes of BC patients. The LDHC level in serum and exosomes could distinguish BC cases from healthy individuals based on their AUCs of 0.9587 and 0.9464, respectively. Besides, the LDHC level in exosomes of BC patients associated with tumor size, and positively correlated with HER2 and Ki-67 expressions (all with P < 0.05). Serum and exosomal level of LDHC negatively correlated with medical treatment and positively with the recurrence of BC. Survival analysis showed that LDH-C4 expression negatively correlated with BC prognosis. Conclusion: Serum and exosomal LDHC may be an effective indicator for the diagnosis, efficacy evaluation, and monitoring the recurrence of BC. LDH-C4 may act as a biomarker that predicts BC prognosis.

Key words: lactate dehydrogenase C4, breast cancer, serum, exosome, diagnosis, prognosis

Funding This study was sponsored by the National Natural Science Foundation of China (Grant number: 81802631), Fujian Provincial Health Technology Project (Grant number: 2016-ZQN-17), and Joint Funds for the Innovation of Science and Technology, Fujian province (Grant number: 2017Y9073), and Science and Technology Program of Fujian Province, China (Grant number: 2018Y2003).

Declaration of interest None

1. Introduction

BC is one of the most common malignant tumors mainly appearing in females worldwide [1]. According to the latest cancer epidemiology in China, BC has been the fourth-most fatal cancer that mostly occurs in female patients with malignancy [2]. Early diagnosis, timely treatment and accurate prognosis are crucial determining factors for BC survival rate, which requires identification of novel tumor markers for the disease as the first imperative for researchers. CTA, expressed only in the germinal epithelium of the testes and some tumor tissues, may be a marker for tumor diagnosis and immunotherapy [3-6]. LDH-C4 protein (also known as LDHX), encoded by the LDHC gene, is expressed merely in

the testes and tumors [7, 8]. The human LDHC gene is located on chromosome 11p15.3-15.5. The full-length mRNA of LDHC is 1179 bp. The LDHC gene has two transcripts with an open reading frame containing 999 base pairs. The LDHC gene encodes a C subunit with a relative molecular mass of 35 kDa. Four identical C subunits undergo polyhomologation to form the catalytically active tetrameric protein, LDH-C4 zymoprotein [6]. As a CTA molecule, the expression of LDH-C4 is specific to mature testes and sperm of healthy males [7, 8]. Specifically, LDH-C4, as an important molecular index for maturation of male germ cells and assessment of fertility, plays a critical role in sperm energy metabolism [7, 9, 10]. Splice variants of LDHC have been observed in the testes of various mammals according to recent researches, often accompanied by mutations such as deletions of exons 5, 4, and 6 [11]. LDH-C4 mRNA and protein are expressed in the myocardium, liver, lung, kidney, brain, skeletal muscle and other tissues of plateau pikas [12, 13]. The expression of LDH-C4 in somatic cells of plateau pikas suggests that it may correlate with energy metabolism. However, few studies have shown solicitous attention on its role in malignant tumors. One study reports that LDHC mRNA is expressed in many types of tumors, and the positive expression rates of its spliceosome are 47% in lung cancer, 44% in melanoma, 35% in BC, and 15% in colon cancer [14]. It can be hypothesized that LDHC may be involved in the onset and development of these cancers. Grunwald et al. present a 25% increase in the mRNA expression of LDHC in non-small-cell lung carcinoma (NSCLC) tissue. Furthermore, the mRNA expression of LDHC in NSCLC was comparable to that in

adenocarcinoma and squamous cell carcinoma [15]. The mRNA and protein expressions of LDHC significantly increase in renal cell carcinoma, and LDHC-positive patients usually associates with worse outcomes [16]. In our previous work, we have developed a novel histochemical staining method for the detection of LDH-C4 activity [17], observing abnormal expression of LDH-C4 in MDA-MB-231 cells [18]. The LDH-C4-specific inhibitor, N-propyl oxamate, can reduce the invasion and migration of MDA-MB-231 cells [18]. In the present study, we investigated serum and exosomal level of LDHC mRNA, quantified the protein expression of LDH-C4 in BC tissues using high-throughput tissue microarray analysis and immunohistochemistry, and evaluated the correlation of LDH-C4 expression with clinical pathological characteristics and BC prognosis.

2. Materials and methods 2.1. Clinical Data Serum samples were collected from 75 BC patients who were admitted to Fujian Cancer Hospital from December 2018 to May 2019, and 120 healthy controls. The BC cases encompassed 24 preliminary diagnosis / preoperative cases, 32 postoperative cases and 19 recurrence cases. All the 75 cases had invasive ductal carcinoma, and were diagnosed by pathological examinations. Collection of all serum samples was approved by the Ethics Committee of Fujian Cancer Hospital (ethical approval certificate: No. SQ2018-015-01). LDH-C4 protein results were obtained from a database using a commercial high-throughput tissue microarray (HBreD145Su02;

SHANGHAI OUTDO BIOTECH, China), which involved 145 patients diagnosed with BC from August 2004 to December 2008 and all were females. The patients aged from 33 to 88 years old, with a median age of 57. Diagnoses were confirmed via pathological analysis, and none of the patients had received preoperative radiotherapy and chemotherapy. Among the 145 females, 1 had intraductal carcinoma, 142 invasive carcinoma (including invasive ductal carcinoma and lobular carcinoma), and 2 mucinous carcinoma. All samples were collected from primary organs with no definite signs of distant organ metastasis. Sixty-nine patients had lymphatic metastasis, and 74 had no lymphatic metastasis. Based on the 6th Edition of the AJCC breast cancer TNM staging system, and the clinical staging of the BC patients in our study was as follows: 1 in stage 0, 22 in stage I, 75 in stage II, and 39 in stage III (information of TNM staging in 5 cases was missing). The high-throughput tissue microarray (HBre-Duc090Sur-01, 90 cases; SHANGHAI OUTDO BIOTECH, China) was utilized as the paracancerous control.

2.2. Vesicle isolation, identification and total RNA extraction Serum-derived exosomes / vesicles were firstly purified according to the protocol of the exoRNeasy Serum / Plasma Midi Kit (QIAGEN, Catalog No.77044) (Part I: vesicle isolation). Briefly, 1 mL serum sample was prepared for serum vesicle isolation, and detailed protocols were documented in www.qiagen.com/HB-1179. The isolated exosomes from serum were identified and photographed by the transmission electron microscope (FEI TECNAI G2, Philips) with an accelerating voltage of 80 kV.

Moreover, the exosome marker proteins, CD9 and CD 63, were further detected by immunoblotting. The primary antibodies and the dilutions included: rabbit monoclonal [Abcam, EPR2949] to CD9 (1:500), rabbit monoclonal [Abcam, EPR21151] to CD63 (1:800), and mouse monoclonal anti-human β-actin (Beyotime, 1:1000). A detailed protocol for immunoblotting was described in our previous study [18]. Total RNA was extracted from serum and serum-derived exosome according to the procedures of the miRNeasy Kit (QIAGEN, Catalog No. 217184) and the exoRNeasy Serum / Plasma Midi Kit (QIAGEN, Catalog No.77044) (Part II: RNA isolation). Detailed protocols were documented in www.qiagen.com/HB-1002 and www.qiagen.com/HB-1179.

2.3.Quantitative real-time PCR (qRT-PCR) Total RNA was reverse-transcribed to cDNA using a Transcriptor First Strand cDNA synthesis kit (Roche) according to the manufacturer’s instructions. The product was amplified by SYBR Green Master (ROX) using the following primer sequences: LDHC-F:

5'-TCATTCCTGCCATAGTCCA-3',

5'-CAATTACACGAGTTACAGGTA-3'; 5'-TCGACAGTCAGCCGCATCTTCTTT-3';

LDHC-R: GAPDH-F:

and

GAPDH-R:

5'-ACCAAATCCGTTGACTCCGACCTT-3'. Quantitative RT-PCR was performed using an ABI7500 qRT-PCR detector under the following conditions: 40 cycles of denaturation at 95°C for 10 min, followed by 95°C for 15 s, with extension at 60°C

for 1 min. The 2-ΔΔCT method was adopted for relative quantification.

2.4.Immunohistochemical analysis High-throughput breast cancer tissue microarray (HBreD145Su02, each tissue microarray

contained

145

points

of

BC

tissues)

was

selected

for

immunohistochemical analysis. Paraffin-embedded tissues / cells were fixed and analyzed using an EnVision DAB Assay Kit in EliVisionTM Plus two-step detection system, with rabbit monoclonal anti-human LDHC primary antibody (Abcam, Catalog. No. ab52747, 1:100) as the primary antibody. Phosphate buffer solution (PBS) was utilized as a negative control. The experimental results were explained by an experienced pathologist in clinic pathological analysis. A detailed protocol was described in our previous study [18].

2.5. Statistical analysis All data were analyzed using SPSS 16.0 software. Measurement data were presented as means ± SD. When normality and homogeneity of variance were satisfied, Student’s t-test was adopted to compare differences between the two groups. χ2 test and Spearman correlation analysis were performed to analyze the correlation between LDH-C4 expression and clinical pathological characteristics. Univariate survival analysis was performed using the Kaplan-Meier method to generate a survival curve. A P value of less than 0.05 was accepted as statistical significance.

3. Results 3.1 Identification of the isolated vesicles The purified exosomes were first identified by the transmission electron microscope. As shown in Figure 1A, the membranous vesicles, 40 to 140 nm in diameter, were observed in eluent. Immunoblotting showed that the exosome marker proteins, CD9 and CD 63, were datable in the purified eluent (Figure 1B), suggesting that serum-derived exosomes were successfully isolated. 3.2 Serum and serum-derived exosome LDHC was an appropriate biomarker for BC Quantitative RT-PCR indicated that the positive rates of LDHC mRNA in serum and serum-derived exosomes of BC patients were 91.66% (22/24) and 87.50% (21/24), while those in healthy controls were only 9.16% (11/120) and 6.67% (8/120). Relative quantification showed that the average expression level of serum LDHC in newly diagnosed patients was 9.79 times higher than that of healthy controls (Figure 2A, B). The level of exosomal LDHC in BC patients was 8.87 times higher than that of healthy controls (Figure 2 D,E). Receiver operating characteristic (ROC) curve analysis revealed that the sensitivity, specificity and AUC of serum LDHC for discerning BC patients from healthy controls were 90.91%, 86.36% and 0.9587, respectively (Youden index = 0.77) (Figure 2C). The sensitivity, specificity and AUC of exosomal LDHC for identifying the two groups were 87.50%, 90.48% and 0.9464 (Youden index = 0.78) (Figure 2F). These indicated that serum and exosomal LDHC might act as desirable indexes for the diagnosis of BC.

3.3 Correlation between the LDHC expression in serum and serum-derived exosome and clinical pathological characteristics of BC patients Our correlation analysis revealed that the expression of serum LDHC positively correlated with the levels of Ki-67 (r2 = 0.6381, P < 0.0001) and HER2 (r2 = 0.6987, P < 0.0001). Furthermore, LDHC expression in serum-derived exosome also associated with BC tumor size (P = 0.044), level of Ki-67 (r2 = 0.3820, P = 0.0022) and HER2 expression (r2 = 0.6497, P < 0.0001). Serum and exosomal LDHC did not correlate with age, pathological grading, lymphatic metastasis and other clinical pathological indices, such as estrogen receptor (ER), and Cerb-1 (all with P > 0.05) (Figure 3 and Table 1).

3.4 Diagnostic values of serum LDHC in preoperative, postoperative and recurrence BC patients We determined the LDHC expression in serum and exosome varied in preoperative, postoperative and recurrence BC patients. The level of serum LDH-C4 in postoperative patients was lower than that in preoperative ones, whereas the expression of serum LDHC in recurrent patients was higher than that in both pre- and post-operative patients (Figure 4A, 4B). Similarly, the level of exosomal LDHC in preoperative patients was significantly higher than that in postoperative ones. But in recurrent patients, the level of exosomal LDHC was elevated (Figure 4C, 4D). ROC analyses revealed that serum LDHC distinguished preoperative patients from postoperative or recurrent ones based on AUCs, of 0.8491 and 0.7584 respectively

(Figure 4E, 4F), whereas exsomal LDHC yielded AUCs of 0.8173 and 0.6986 respectively (Figure 4G, 4H). All data above indicated that serum and exosomal LDHC might be a promising marker for effectively monitoring and predicting the recurrence of BC.

3.5 LDH-C4 was up-regulated in BC tissues and was a promising diagnostic and prognostic biomarker Immunohistochemistry analysis showed that LDH-C4 was primarily expressed in the cytoplasm of BC cells, and to a lesser extent in nuclei (Figure 5A). Of the 142 patients (3 cases were absent) who underwent the analysis, 130 were positive for LDH-C4 expression, and the rate of positive LDH-C4 expression in BC tissues was 91.55%. Among LDH-C4 positive samples, 33.10% presented low expression (-/+) and 66.90% high expression (+/++). However, Low expression of (-/+) LDH-C4 occurred only in 10% (9/90) of adjacent normal tissues, and 90% of the adjacent normal tissue samples were negative for LDH-C4. Furthermore, LDH-C4 level was elevated in patients with stage III compared with those in stage 0-II (Figure 5B). Correlation analysis showed that LDH-C4 expression showed no correlations with age, tumor size, TNM staging, presence of lymphatic metastasis or pathological grading (all with P > 0.05) (Table 2). Survival analysis revealed that LDH-C4 expression negatively correlated with BC prognosis, and the 10-year survival rate of LDH-C4-negative BC patients was significantly higher than that of LDH-C4-positive BC patients (Figure 5C). Furthermore, the BC patients with strongly positive

expression (+++) of LDH-C4 presented much shorter overall survival time than those with merely positive expression (++/+) of LDH-C4 (Figure 5D), which suggested that LDH-C4 might serve as a promising prognostic indicator for patients with BC.

4. Discussion CTA is only expressed in the germinal epithelium of the testes and some tumor tissues [3-6]. LDH-C4, a class of LDH isozyme and a component of the lactate-pyruvic acid transition in carbohydrate metabolism pathways, specifically expressed in sperm and testicular tissue of birds and mammals [5, 7, 10]. As a class of CTA, LDH-C4 is expressed in some malignant tumor cells and translated to zymoprotein with normal biological activity [7, 15, 18]. Recently, studies have reported that LDH-C4 is highly expressed in lung, breast and kidney carcinomas, melanoma and many other malignant tumors [14-16, 18]. In this study, we determined the serum, serum-derived exosomal and tissue expression levels of LDH-C4 in BC patients, and evaluated the diagnostic and prognostic value of LDHC / LDH-C4 in BC. Studies have shown that tumor patients present higher level of free circulating nucleic acids than healthy individuals [19, 20]. In the present study, we report for the first time that LDHC mRNA is expressed in the serum of BC patients, with a positive rate of 91.66%. Furthermore, high percentages of diagnostic sensitivity and specificity of this marker are presented. Exosomes, 40 nm to 100 nm in diameter, are a class of extracellular vesicles (EV) released from cells [21]. Exosomes consist of protein, DNA and RNA, and prevent these biomolecules from degradation in the blood,

allowing for detection in the tumor microenvironment [22]. Emerging evidence has suggested that exosomes can regulate angiogenesis, immunity and metastasis, playing a significant role in tumor development [23]. Circulating molecules in exosomes may serve as non-invasive biomarkers for early detection of tumors, diagnosis and treatment monitoring [24, 25]. This study is the first to report the expression of LDHC in the serum-derived exosomes of BC patients. The two forms of LDHC (in serum and exosomes) are both promising markers for diagnosis, efficacy evaluation, and repeated monitoring of BC. However, exosomal LDHC only yields an AUC of 0.6986 in predicting BC recurrence, a less powerful and effective figure. Thus, more evidences are still needed. Koslowski et al. show that the positive rate of LDHC mRNA in BC tissues is 35% [14]. We found that the positive rate of LDH-C4 protein in BC tissues was 91.5%. In addition, our survival analysis revealed that the survival rate of LDHC-positive BC patients was much lower than that of LDHC-negative BC patients. We also found that the levels of LDHC negatively associated with the survival rate of BC patients. These results indicate that LDH-C4 is a potential important index for determination of BC prognosis. A recent study has reported that the mRNA and protein expressions of LDHC are significantly up-regulated in renal cell carcinoma with poorer prognoses in LDHC-positive patients [16], which is consistent with our results. Both the role of LDH-C4 in the onset and development of tumors and the underlying mechanism behind it remain unclear. Nevertheless, Wang et al. have

reported the expressions of LDHC mRNA and LDH-C4 protein in the myocardium, liver, lung, kidney, brain, skeletal muscle and other tissues of plateau pikas [12]. LDH-C4 expression in somatic cells of plateau pikas probably connects with energy metabolism. As growth and metabolism of tumor cells is energy-intensive, studies have shown that ATP can be generated by glycolysis even in the absence of oxygen [26]. Chen et al. have detected the level of LDHC in osteosarcoma, and have confirmed its role in glycolysis in tumors [27]. In conclusion, our study investigated the expression of LDHC in the serum, exosomes and BC tissues of the selected patients. The results indicate that LDHC / LDH-C4 is a promising biomarker for diagnosis, efficacy evaluation, monitoring BC recurrence and predicting the prognosis for BC in clinical practice. We aspire that our study of LDHC/LDH-C4 will shed light on further application of this molecular marker in BC. However, because of the small sample size in our study, further investigations are required.

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Figure legends Figure 1. Identification of isolated vesicles in serum. (A) isolated exosomes from serum were photographed by the transmission electron microscope. (B) Expression of the exosome marker proteins, CD9 and CD 63, were detected by immunoblotting.

Figure 2. Diagnostic value of serum and exosome LDHC in BC. (A, B) Quantitative RT-PCR results showed that serum LDHC was present and up-regulated in most BC case compared with healthy individuals. (C) Receiver operating characteristic (ROC) curve of serum LDHC for confirmation of BC. (D, E) qRT-PCR showed positive expression of LDHC in serum sourced exosomes. (F) ROC curve of serum sourced exosome LDHC in detecting BC.

Figure 3. Correlation of serum and serum sourced exosome LDHC expression with pathological characteristics. Correlation of serum LDHC expression with (A) Ki-67, (B) HER2, (C)ER and (D) PR was assessed. Serum sourced exosome LDHC expression was also found to be associated with (A) Ki-67 and (B) HER2 expression, but was not related to (C) ER and (D) PR levels.

Figure 4. Comparison of serum and exosome LDHC expression in pre-operative, post-operative and recurrence patients with BC. Relative (A,B) serum and (C,D) exosomal LDHC expression levels in pre-operative, post-operative and recurrence patients. ROC curves of serum LDHC in discriminating pre-operative cases from (E) post-operative and (F) recurrence cases. And ROC curves of exsome LDHC in detecting pre-operative cases from (G) post-operative and (H) recurrence cases. **P<0.01.

Figure 5. Up-regulation of LDH-C4 in BC tissues. (A) Immunohistochemistry showed that LDH-C4 expression was higher in BC tissues; normal testis tissue was utilized as an LDH-C4 positive control, 400×. (B) LDH-C4 expression was higher in patients with stage 0-II BC than those with stage III. (C) Kaplan-Meier analysis shows LDH-C4 expression was negatively correlated with BC prognosis, and (D) higher levels of LDH-C4 resulted in worse prognosis.

Table 1. Clinical characteristics of serum and exosomal LDHC mRNA expression in BC

Serum LDHC Clinicopathological features

Exosomal LDHC

Positive

Negative

P

Positive

Negative

P

(n=22)

(n=2)

value

(n=21)

(n=3)

value

Age (years)

0.174

0.089

≤50

11

2

10

3

>50

11

0

11

0

0.108

Tumor size (cm)

0.044

≤2

9

2

8

3

>2

13

0

13

0

TNM stage

0.253

0.151

Stage 0-II

13

2

12

3

Stage III

9

0

9

0

Lymphatic metastasis

0.140

0.064

Yes

12

0

12

0

No

10

2

9

3

Table 2. Clinical characteristics of LDH-C4 expression in BC tissue. Total case Clinicopathological features

size

LDH-C4 expression (-/+)

LDH-C4 expression

P value

(++/+++)

Age (years)

0.717

≤50

39

12

27

>50

103

35

68

Histological grade

0.171

I-II

4

1

3

II

70

27

43

II-III

50

17

33

III

18

2

16

Tumor size (cm)

0.810

≤2

36

8

28

>2

102

39

63

TNM stage

0.970

Stage 0-II

98

33

65

Stage III

39

13

26

Lymphatic metastasis

0.186

Yes

69

19

50

No

71

27

44

Highlights 1. LDHC mRNA and protein are up-regulated in breast cancer. 2. Serum and exosomal level of LDHC may be an effective indicator for the diagnosis, efficacy evaluation, and monitoring the recurrence of breast cancer. 3. LDH-C4 serves as a promising prognostic indicator for breast cancer.

Authors contribution Z. Cui acquired, analyzed, and interpreted the data and drafted, revised, and finally approved the article; Y. Chen, M. Hu, Y. Lin, Shu. Zhang acquired, analyzed, and interpreted the data and finally approved the article; L. Kong, and Y. Chen, conceived and designed the study and critically revised and finally approved the article.