Plasma TuM2-PK correlates with tumor size,CRP and CA 15-3 in metastatic breast carcinomas;short versus long term follow up study of the Egyptian breast cancer patients

Abstract

BACKGROUND

: The key regulator of tumor metabolome is the glycolytic isoenzyme M2-PK which favors the generation of nucleic acid via glutaminolysis as hypoxic adaptive mechanism in the tumor cells.

AIM

: The study aimed to evaluate the prognostic role of M2-PK, CRP, and CA 15-3 in preoperative and metastatic breast carcinomas.

PATIENTS AND METHODS

: The study included 70 females; 15 controls, 33 preoperative primary breast carcinomas clinically metastasis free, and 22 clinically diagnosed metastatic breast carcinomas. M2-PK and CA 15-3 were detected by ELISA. CRP was quantified using the CRP LATEX kit.

RESULTS

: TuM2-PK significantly increased in metastatic and preoperative groups when compared to controls ( $p=$ 0.049, $p=$ 0.001); respectively. Both CRP and CA 15-3 were significantly increased in metastatic than the preoperative group ( $p=$ 0.002). CA 15-3 was significantly increased in both groups when compared to controls ( $p=$ 0.016; $p<$ 0.001; respectively). TuM2-PK level significantly related to tumor size in metastatic group ( $p=$ 0.006) and with menstruation status ( $p=$ 0.039), and liver metastasis ( $p=$ 0.036) in preoperative group. TuM2-PK significantly correlated with CRP ( $r=$ 0.793, $p=$ 0.004), and CA 15-3 ( $r=$ 0.568, $p=$ 0.006) in the metastatic group.

Metastatic group with TuM2-PK $\leqslant$ 15 U/ml had significantly higher survival rate than those with $>$ 15 U/ml ( $\chi^{2}=$ 13.841, $p<$ 0.001) within 3.3–4.2 but not after 10–20 years follow up period. Metastasis to bone and lymph nodes significantly increased in the metastatic than the preoperative group ( $p=$ 0.002, $p=$ 0.013; respectively). Within 3.3–4.2 years, CA15.3 has the highest prognostic performance in metastatic group while both TuM2-PK and CRP have same specificity. On the other hand, TuM2-PK has the highest prognostic performance in preoperative group. After 20 years follow up period, there was neither significant difference in the performance of the three markers in predicting mortality in metastatic and preoperative groups nor in predicting metastasis in preoperative group.

CONCLUSION

: Current results document for the first time, a cross-talk between TuM2-PK and each of CRP and CA 15-3 in metastatic breast cancer.

Keywords

Breast carcinoma CA 15-3 CRP immunity inflammation prognostic performance TuM2-PK tumor markers

1. Introduction

1.1 TuM2-PK

Pyruvate Kinase (PK; (EC 2.7.1.40) is a cytosolic glycolytic enzyme which catalyzes the formation of pyruvate and ATP from phosphoenol pyruvate (PEP) and ADP. It exists in four isoenzyme forms (L, R, M1, and M2). M2-PK is the only detectable isoenzyme in early fetal tissues and proliferating cells. During development, it is gradually replaced by L, R, and M1 types. During tumorigenesis, the tissue specific PK isoenzymes generally disappear and M2-PK is expressed [1, 2]. M2-PK exists in two forms; the active tetrameric form with high affinity and the less active dimeric form with low affinity to its substrate; PEP. The ratio between the two forms determine glucose fate in the cell.

Table 1
Age, tumor prognostic factors, and patients ‘outcomes of the studied groups

Parameters	Control ( $n=$ 15)	Preoperative ( $n=$ 33)		Metastatic ( $n=$ 22)
Age (years) ${}^{+}$
Min–Max	32–60	34–76		27–70
M $\pm$ SD	43.07 $\pm$ 9.15	51.06 $\pm$ 11.24*^		44.86 $\pm$ 11.16*
Median	40	49		44.5
Menopausal status; $n$ (%)
Premenopausal	11 (73.3)	17	(51.5)	15	(68.2)
Postmenopausal	4 (26.7)	16	(48.5)	7	(31.8)
Tumor histological type
Invasive ductal carcinoma (IDC);	–	30	(90.90)	22	(100)
not otherwise specified (NOS)
Metastatic carcinoma/Paget’s disease	–	1	(3.03)	0	(0)
Intraductal cribiform carcinoma	–	1	(3.03)	0	(0)
Invasive papillary carcinoma	–	1	(3.03)	0	(0)
ER; $n$ (%)
Negative	–	6	(18.2)	10	(45.5)
Positive
$+$	–	8	(24.2)	1	(4.5)
$++$	–	11	(33.3)	6	(27.3)
$+++$	–	8	(24.2)	5	(22.7)
PR; $n$ (%)
Negative	–	6	(18.2)	10	(45.5)
Positive
$+$	–	8	(24.2)	4	(18.2)
$++$	–	6	(18.2)	3	(13.6)
$+++$	–	12	(36.4)	5	(22.7)
LNM; $n$ (%)
Negative	–	6	(18.2)	1	(4.5)
Positive	–	27	(81.8)	21	(95.5)
Number of the involved LNs; $n$ (%)
0	–	7	(21.2)	1	(4.5)
1 (1–3)	–	9	(27.3)	3	(13.6)
2 (4–9)	–	14	(42.4)	12	(54.5)
3 ( $>$ 10)	–	3	(9.1)	6	(27.3)
Tumor grade; $n$ (%)
II	–	29	(87.9)	16	(72.7)
III	–	4	(12.1)	6	(27.3)
Tumor size (cm); $n$ (%)
$<$ 2	–	1	(3.03)	3	(13.6)
2–4	–	24	(72.72)	9	(40.9)
$>$ 4	–	6	(18.18)	7	(31.81)
Unknown (excision biopsy)	–	2	(6.06)	3	(13.6)
Tumor stage; $n$ (%)
I	–	1	(3)	0	(0)
II	–	10	(30)	0	(0)
III	–	22	(66.6)	0	(0)
IV	–	0	(0)	22	(100)
Patients’ outcomes (after 20 years follow up)
Metastasis	–	15	(45.45)	22	(100)
Death	–	25	(96.2)	16	(94.1)
NPI ${}^{{47}}$
Excellent (2.02–2.4)	–	0	(0)	0	(0)
Good (2.4–3.4)	–	4	(12.12)	0	(0)
Moderate 1 (3.41–4.4)	–	10	(30.30)	5	(22.72)
Moderate 2 (4.41–5.4)	–	10	(30.30)	6	(27.27)
Poor (5.41–6.4)	–	7	(21.21)	8	(36.36)

Table 1, continued
Parameters	Control ( $n=$ 15)	Preoperative ( $n=$ 33)		Metastatic ( $n=$ 22)
NPI ${}^{{47}}$
Very poor (6.41–6.8)	–	0	(0)	0	(0)
Unknown (due to unknown tumor size)	–	2	(6.06)	3	(13.63)

${}^{+}$ $F=$ 3.726, $p=$ 0.029 (statistically significant at $p\leqslant$ 0.05). ${}^{*}$ Significant when comparing between metastatic and preoperative groups. ${}^{\#}$ Significant when comparing between metastatic and control groups. ${}^{\wedge}$ Significant when comparing between preoperative and control groups. $n$ : number; M $\pm$ SD: Mean $\pm$ Standard deviation; ER: estrogen receptor; PR: progesterone receptor; $F$ : value for ANOVA test; NPI: Nottingham prognostic index.

Since the tumor cells display a change in the cellular metabolic demands, they require the less active dimeric form of M2-PK. Therefore, M2-PK becomes the dominant expressed form in the tumor, released into the circulation, and thus it is called TuM2-PK. The dissociation of the tetrameric form into the dimeric form is induced by interaction with oncoproteins, cellular phosphotyrosine signaling, and cellular regulator metabolites [3, 4, 5, 6, 7, 8, 9, 10]. This shift ensures efficient ATP synthesis and a high capacity for the biosynthesis of metabolites which are vital for the proliferation, growth and survival of tumor cells under limited oxygen and nutrient supply [11, 12, 13, 14].

Limited data is available for TuM2-PK in breast cancer. However, in the metastasized breast cancer, plasma M2-PK was suggested to be a helpful tool in monitoring the therapeutic success [15, 16, 17].

1.2 C-reactive protein (CRP)

It is evident now that there is an association between cancer and inflammation [18]. CRP is an acute phase short pentraxin protein which is synthesized in hepatocyte and released into the circulation in response to inflammation, trauma, tissue damage, and cancer [19, 20, 21, 22]. The circulatory form is a pentameric isoform which increases in proportion to the degree of tissue damage. Association between breast cancer and inflammation was suggested as the seventh hallmarks of cancer [23]. Leucocytes invade breast cancer and release interleukin-6 (IL-6); the main inducer of CRP synthesis. IL-6 helps CRP in acting as opsonin leading to tumor cell lysis via aiding in binding CRP to phospholipids on tumor cells and activation of classic C1q complement pathway. In animal model, a link between CRP and breast cancer invasion was elucidated via upregulated expression of sphingosine-1-phosphate (S1P) which in turn triggers transcriptional activation of matrix metalloproteinase-9 through reactive oxygen species, calcium ions, and c-fos. Regardless the contradictory results of linkage between CRP and breast cancer risk, an elevated level of CRP at the time of diagnosis indicates the aggressiveness of the breast tumor, high risk of recurrence, and metabolic disturbance. It also acts as a predictor of cardiotoxicity in patients treated with chemotherapy [24, 25].

1.3 Cancer antigen or carbohydrate antigen 15-3 (CA 15-3)

CA 15-3 detects the soluble moiety of the transmembrane mucin 1 protein which belongs to a large family of glycoproteins with high molecular weight and heterogeneously expressed on the apical surface of normal epithelial cell types, including those of the breast [26, 27]. The extracellular domain of mucin 1 can be released by ADAM17 and MT-MMP1 proteases into the extracellular matrix and thus shed into serum [28, 29]. CA 15-3 is considered the most routine marker for monitoring breast cancer. CA 15-3 level is elevated in patients with malignant disease more than benign diseases [30, 31]. CA 15-3 is elevated in a proportion of breast cancer patients with distant metastases [32, 33]. Though current American Society of Clinical Oncology and National Comprehensive Cancer Network guidelines do not recommend its use for surveillance purposes, elevated CA 15-3 is used to anticipate detection of recurrences in patients with breast cancer and as an additional tool in evaluating therapeutic response of advanced disease [34]. Preoperative levels of CA 15-3 have a significant and independent relation to outcome in patients with early breast cancer [35, 36]. Patients with high concentrations have a significantly worse prognosis than those with low concentrations, both in terms of disease-free survival (DFS) and overall survival (OS), probably due to a larger burden of occult disease [37, 38, 39, 40, 41, 42]. Patients with locoregional disease alone and increased CA 15-3 levels require regular screening for systemic dis- ease [43].

The current study was designed to evaluate the prognostic value of plasma TuM2-PK marker in metastasized breast cancers in combination with the well-known CA 15-3 (as a routine tumor marker) and the inflammatory marker; CRP. The additive value of the three markers in diagnosing the increasing burden of breast cancer metastasis was also investigated.

Table 2
TuM2-PK among the studied groups

TuM2-PK (U/ml)	Metastatic ( $n=$ 22)		Preoperative ( $n=$ 33)		Control ( $n=$ 15)		Test of sig.	$p$
	$N$	%	$N$	%	$N$	%
$\leqslant$ 15	11	50.0	8	24.2	14	93.3	$\chi^{2}=$ 19.861 ${}^{*}$	$<$ 0.001 ${}^{*}$
$>$ 15	11	50.0	25	75.8	1	6.7
Sig. bet. groups	$p_{1}=$ 0.049 ${}^{}$ , $p_{2}=$ 0.011 ${}^{}$ , $p_{3}<$ 0.001 ${}^{*}$
Min–Max	6.50–180.0		7.0–200.0		6.50–16.0		${}^{\rm{KW}}\chi^{2}=$ 10.816 ${}^{*}$	0.004 ${}^{*}$
Mean $\pm$ SD	42.0 $\pm$ 53.32		36.50 $\pm$ 38.44		11.77 $\pm$ 2.93
Median	16.50		27.0		11.50
Sig. bet. groups	$p_{1}=$ 0.429, $p_{2}=$ 0.049 ${}^{}$ , $p_{3}<$ 0.001 ${}^{}$

$\chi^{2},p$ : $\chi^{2}$ and $p$ values for Chi square test for comparing between the different groups. ${}^{\rm{KW}}\chi^{2}$ : Chi square for Kruskal Wallis test of significance between groups was done using Mann Whitney test. $p_{1}$ : $p$ value for comparing between Metastatic and Preoperative groups. $p_{2}$ : $p$ value for comparing between Metastatic and control groups. $p_{3}$ : $p$ value for comparing between Preoperative and control groups. *: Statistically significant at $p\leqslant$ 0.05. Sig: significance; bet: between.

Table 3

Relation between TuM2-PK and different parameters in each group

	TuM2-PK (U/ml)
	Metastatic				$p$	Preoperative				$p$
	$\leqslant$ 15 ( $n=$ 11)		$>$ 15 ( $n=$ 11)			$\leqslant$ 15 ( $n=$ 8)		$>$ 15 ( $n=$ 25)
Age (years)	47.5 $\pm$ 11		42.3 $\pm$ 11.2		0.287	47 $\pm$ 7.9		52.4 $\pm$ 12		0.247
Tumor size (cm)	2	(1.5–5)	4.5	(2.5–10)	0.006 ${}^{{*}}$	3.8	(2–9)	3.7	(1.5–8)	0.520
Involved LN%	66.7	(37.5–100)	68.1	(43.8–100)	0.458	45.5	(0–75)	66.7	(0–100)	0.314
ER
–ve	4	(36.4%)	6	(54.5%)	0.859	1	(12.5%)	5	(20%)	1.000
$+$	1	(9.1%)	0	(0%)		2	(25%)	6	(24%)
$++$	3	(27.3%)	3	(27.3%)		3	(37.5%)	8	(32%)
$+++$	3	(27.3%)	2	(18.2%)		2	(25%)	6	(24%)
PR
–ve	4	(36.4%)	6	(54.5%)	0.267	2	(25%)	5	(20%)	0.813
$+$	1	(9.1%)	3	(27.3%)		1	(12.5%)	7	(28%)
$++$	3	(27.3%)	0	(0%)		2	(25%)	4	(16%)
$+++$	3	(27.3%)	2	(18.2)		3	(37.5%)	9	(36%)
Grade
I	0	(0%)	0	(0%)	0.149	0	(0%)	0	(0%)	0.550
II	6	(54.5%)	10	(90.9%)		8	(100%)	21	(84%)
III	5	(45.5%)	1	(9.1%)		0	(0%)	4	(16%)
LNM
Negative	0	(0%)	1	(9.1%)	1.000	1	(12.5%)	5	(20%)	1.000
Positive	11	(100%)	10	(90.9%)		7	(87.5%)	20	(80%)
Menstruation status
Pre	7	(63.6%)	8	(72.7%)	1.000	7	(87.5%)	10	(40%)	0.039 ${}^{{*}}$
Post	4	(36.4%)	3	(27.3%)		1	(12.5%)	15	(60%)
Site of metastasis
None	0	(0%)	0	(0%)	–	3	(37.5%)	15	(60.0%)	0.418
Liver	2	(18.2%)	4	(36.4%)	0.635	3	(37.5%)	1	(4.0%)	0.036 ${}^{{*}}$
Bone	3	(27.3%)	7	(63.6%)	0.087	1	(12.5%)	2	(8.0%)	1.000
Axillary	3	(27.3%)	3	(27.3%)	1.000	0	(0.0%)	1	(4.0%)	1.000
Lung	2	(18.2%)	4	(36.4%)	0.635	3	(37.5%)	2	(8.0%)	0.078
Breast	6	(54.5%)	1	(9.1%)	0.063	1	(12.5%)	8	(32.0%)	0.394
Brain	0	(0%)	0	(0%)	–	1	(12.5%)	0	(0.0%)	0.242
Outcome
Died	8	(88.9%)	8	(100%)	1.000	6	(85.7%)	19	(100%)	0.269
Alive	1	(11.1%)	0	(0%)		1	(14.3%)	0	(0%)

*: Statistically significant at $p\leqslant$ 0.05. N.B.: 60% of patients with TuM2-PK $>$ 15 U/ml developed metastasis during the 59 months follow up period.

Table 4

Correlation between TuM2-PK, CRP and CA15.3 in metastatic and preoperative groups

		Metastatic			Preoperative
		TuM2-PK	CRP	CA15.3	TuM2-PK	CRP	CA15.3
M2-PK	$r$
	$p$
CRP	$r$	0.793			0.428
	$p$	0.004 ${}^{*}$			0.165
CA15.3	$r$	0.568	0.542		0.120	0.021
	$p$	0.006 ${}^{*}$	0.085		0.506	0.948

*: Statistically significant at $p\leqslant$ 0.05.

Figure 1.

Plasma TuM2-PK and overall survival in the metastatic (1a), preoperative (1b) groups, and disease free survival in the preoperative group (1c) within the initial follow up period (40 months).

Table 5

CA 15-3 among the studied groups

CA15.3 (U/ml)	Metastatic ( $n=$ 22)		Preoperative ( $n=$ 33)		Control ( $n=$ 15)		Test of sig.	$p$
	N	%	N	%	N	%
$\leqslant$ 36	8	36.4	25	75.8	15	100.0	$\chi^{2}=$ 18.255 ${}^{*}$	$<$ 0.001 ${}^{*}$
$>$ 36	14	63.6	8	24.2	0	0.0
Sig. bet. groups	$p_{1}=$ 0.003 ${}^{}$ , $p_{2}<$ 0.001 ${}^{}$ , $p_{3}=$ 0.044 ${}^{*}$
Min–Max	11.0–242.0		0.0–205.0		5.0–28.0		${}^{\rm{KW}}\chi^{2}=$ 20.743	$<$ 0.001 ${}^{*}$
Mean $\pm$ SD	72.41 $\pm$ 70.96		31.02 $\pm$ 36.43		28.0 $\pm$ 14.13
Median	44.0		19.0		7.56
Sig. bet. groups	$p_{1}=$ 0.002 ${}^{}$ , $p_{2}<$ 0.001 ${}^{}$ , $p_{3}=$ 0.016 ${}^{*}$

$\chi^{2},p$ : $\chi^{2}$ and $p$ values for Chi square test for comparing between the different groups. ${}^{\rm KW}\chi^{2}$ : Chi square for Kruskal Wallis test of significance between groups was done using Mann Whitney test. $p_{1}$ : $p$ value for comparing between Metastatic and Preoperative groups. $p_{2}$ : $p$ value for comparing between Metastatic and control groups. $p_{3}$ : $p$ value for comparing between Preoperative and control groups. *: Statistically significant at $p\leqslant$ 0.05.

Table 6

CRP among the metastatic and preoperative groups

CRP (mg/L)	Metastatic ( $n=$ 11)		Preoperative ( $n=$ 12)		Test of sig.	$p$
	No.	%	No.	%
$\leqslant$ 5	0	0.0	3	25.0	$\chi^{2}=$ 3.163	${}^{\rm FE}$ $p=$ 0.217
$>$ 5	11	100.0	9	75.0
Min.–Max.	6.88–88.49		0.24–41.01		${Z}=$ 3.077 ${}^{*}$	0.002 ${}^{*}$
Mean $\pm$ SD.	38.60 $\pm$ 30.66		9.86 $\pm$ 10.72
Median	17.68		6.79

$\chi^{2},p$ : $\chi^{2}$ and $p$ values for Chi square test for comparing between the two groups. FE: Fisher Exact for Chi square test. $Z, p$ : $Z$ and $p$ values for Mann Whitney test for comparing between the two groups. *: Statistically significant at $p\leqslant$ 0.05.

Table 7

Death and metastasis according to the NPI

NPI	Metastasis				Death
	Preoperative ( $n=$ 33)		Metastatic ( $n=$ 22)		Preoperative ( $n=$ 33)		Metastatic ( $n=$ (22)
	$N$ (%)		$N$ (%)		$N$ (%)		$N$ (%)
Good	0	(0)	0	(0)	1	(25)	0	(0)
Moderate 1	6	(60)	5	(100)	9	(90)	3	(60)
Moderate 2	3	(30)	6	(100)	9	(90)	6	(100)
Poor	6	(85.71)	8	(100)	4	(57.14)	5	(62.5)
Unknown	0	(0)	3	(100)	2	(100)	2	(66.66)
Total	15	(45.45)	22	(100)	25	(96.2)	16	(94.1)

2. Patients and methods

Seventy subjects were randomly selected from patients referred to the clinics of cancer management and research department (MRI, Alexandria University, Egypt) from 1996 to 2005. Blood and tissue samples were collected from subjects according to the ethical principles stated in Belmont report [44]. After history taking, clinical examinations, routine laboratory and radiological investigations, and informed consent taking, subjects were subdivided into three main groups:

Control group: Included 15 females with no evidence of any primary or secondary breast cancer.

Preoperative group: Included 33 females with primary breast carcinomas with no clinical evidence of local or systemic metastasis.

Metastatic group: Included 22 females with local and systemic metastatic breast carcinomas.

Patients were treated and followed up as described in El-Abd et al. [45]. Tu M2-PK (ScheBo Biotech, Germany) and CA 15-3 (Can Ag Diagnostic, Sweden) were assayed by a ready to use ELISA kits. CRP was detected using CRP LATEX (BioSystems SA, Spain). Nottingham prognostic index (NPI) was calculated according to the following formula [46]:

NPI $=$ [Lymph node stage [1, 2, 3]] $+$ [Histological grade [1, 2, 3]] $+$ [Tumor size (cm) X (0.2)]

Statistical analysis was performed using IBM SPSS statistics for Windows, version 20 (Armonk, NY: IBM Corp. 2011). Qualitative data were described using number and percent and was compared using Chi square test. Normally, quantitative data was expressed in mean $\pm$ SD and was compared using student t-test. Abnormally, quantitative data was expressed using median Minimum-Maximum (Min.–Max.) and was compared using Mann Whitney test.

3. Results

Age, tumor prognostic factors, and patients’ outcome are shown in Table 1. Age significantly differed in the studied groups ( $p=$ 0.029). Age increased significantly in preoperative when compared with metastatic and control groups ( $p=$ 0.04, $p=$ 0.02; respectively) (Table 1). Metastatic and preoperative groups differed significantly regarding the occurrence of metastasis and site of metastasis (Table 1).

No significant difference was observed among the studied groups regarding the tumor size, tumor grade, lymph node metastasis (LNM), the number of the involved lymph nodes, hormonal receptor status, menstruation status, and patients’ outcome.

3.1 TuM2-PK

Taking the cut off value 15 U/ml (recommended by the manufacturer), TuM2-PK was increased in 50% (11/22), 75.8% (25/33), and 7% of metastatic, preoperative, and control groups; respectively (Table 2). TuM2-PK significantly increased in metastatic and preoperative groups when compared with control ( $p=$ 0.049, $p=$ 0.001; respectively). However, no significant difference was detected in TuM2-PK level between metastatic and preoperative groups (Table 2). TuM2-PK level significantly related to tumor size in metastatic group ( $p=$ 0.006) and with menstruation status ( $p=$ 0.039), and liver metastasis ( $p=$ 0.036) in preoperative group (Table 3). TuM2-PK significantly correlated with CRP ( $r=$ 0.793, $p=$ 0.004), and CA 15-3 ( $r=$ 0.568, $p=$ 0.006) in the metastatic group (Table 4). No significant relation was detected with age.

3.2 CA 15-3

CA 15-3 (cut off value was 36 U/ml, recommended by the manufacturer) increased in 24.2% (8/33), 63.6% (14/22), and 0% (0/15) of preoperative, metastatic, and control groups; respectively (Table 5). CA 15-3 increased significantly in metastatic than preoperative group ( $p=$ 0.002) (Table 5). It is also increased in both preoperative and metastatic groups when compared with controls ( $p=$ 0.016; $p<$ 0.001; respectively) (Table 5). No significant relation was detected with any of the tumor prognostic factors, age, or patients’ outcome.

3.3 CRP

CRP was detected only in patients with elevated TuM2-PK level. Taking the cut off value 5 mg/L (recommended by the manufacturer), CRP was increased in 100% (11/11), 75% (9/12) in metastatic and preoperative groups; respectively (Table 6). CRP was significantly increased in metastatic group than the preoperative group ( $p=$ 0.002) (Table 6). No significant relation was detected with any of the tumor prognostic factors, age, or patients’ outcome as well.

3.4 Clinical outcome

Metastatic group with TuM2-PK $\leqslant$ 15 U/ml had significantly higher survival rate than those with $>$ 15 U/ml ( $\chi^{2}=$ 13.841, $p<$ 0.001; Fig. 1a) within the initial 40–50 months follow up period. In preoperative group, neither the survival ( $\chi^{2}=$ 0.040, $p=$ 0.842) nor the metastatic rate ( $\chi^{2}=$ 1.353, $p=$ 0.245) has shown any significant difference between patients with TuM2-PK value less or more than the cut off value (15 U/ml) (Fig. 1b and c; respectively).

After 20 years follow up period, no significant difference was observed between the metastatic and preoperative groups regarding the death rate. Metastasis to the bone and lymph nodes significantly increased in the metastatic than the preoperative group ( $p=$ 0.002, $p=$ 0.013; respectively). NPI was mainly in moderate 1 and 2 regions for preoperative group while shifted towards moderate 2 and poor regions for metastatic group (Tables 1 and 7). Perhaps the death was related to other morbidities, poor prognosis, and/or drug toxicity.

Figure 2.

ROC curve for TuM2-PK, CRP, and CA 15-3 in metastatic (2a), and preoperative (2b) groups.

3.5 Receiver-operating characteristics (ROC) curve

During the initial follow up period (40–50 months), CA15.3 has the highest prognostic performance in metastatic group [Area Under the Curve (AUC): 0.955; 95% confidence interval: 0.853–1.056; $p=$ 0.000) (Fig. 2a) while both TuM2-PK and CRP have same specificity (AUC: 0.867; 95% confidence interval: 0.721–1.012; $p=$ 0.002). On the other hand, TuM2-PK (AUC: 0.867; 95% confidence interval (CI): 0.723–1.011; $p=$ 0.001) shows the highest prognostic performance than CRP (AUC: 0.713; 95% CI: 0.515–0.911; $p=$ 0.056) and CA15.3 (AUC: 0.577; 95% CI: 0.359–0.794; $p=$ 0.49) in preoperative group (Fig. 2b). After 20 years follow up period, no significant difference was detected in the prognostic performance of TuM2-PK, CA 15-3, and CRP in predicting mortality in metastatic and preoperative groups. Also no significant difference was observed in the prognostic performance of the TuM2-PK, CA 15-3, and CRP in predicting metastasis in preoperative group.

4. Discussion

The current study documents for the first time a relation between TuM2-PK and each of CA 15-3 and CRP in metastatic breast carcinomas. A cross-talk between M2-PK and mucin 1 was suggested to promote metabolic reprogramming of cancer cells to satisfy the energy/biosynthetic requirements for their growth and survival. Mucin1 master transcriptional reprogramming of metabolic genes including M2-PK and promotes autophagy (self-eating pathway) to maintain survival of cancer cells under hypoxic and limited nutrients environment [48]. Tumor cell autophagy either eradicate tumor cell via increasing the immunogenicity of cancer cells or help them to evade immune system.

Autophagy is also implicated in the regulation of multiple immunological functions (including control of antigen presentation and modulating lymphocyte homeostasis) and influences inflammation, infection and immunity [49].

Recent evidences also implicated M2-PK as a critical regulator of immune cell metabolism and supported its potential role in the genesis of inflammation [50]. It has been shown that M2-Pk boost IL-6 transcription; the main inducer of CRP gene; via phosphorylation of signal transducer and activator of transcription 3 (STAT3) in activated coronary macrophages [51].

The present study further supports the relation between TuM2-PK and tumor size. It has been suggested that metabolic activity or redox-capacity may link M2-PK with cell size-controlling system [52]. Recently, the expression of M2-PK has shown to significantly associate with tumor size ( $p=$ 0.045) in triple negative breast cancer (TNBC) [53].

TuM2-PK was normal in controls except in one case (16 U/ml). Although this value could reflect other diseases that increase the level of M2-PK (such as diabetic nephropathy or rheumatic diseases or hyperlipidaemia or polytruma) [54, 55]; that are not investigated in the current study, this value would also be considered normal since other studies selected other values to achieve better sensitivity and specificity of M2-PK. Landt et al. [56] found that 16 U/ml is a better cut off value which enables achieving higher test specificity. While, Kaura et al. [57] chose the cut off value 17 U/ml to achieve discrimination between malignant and non-malignant lesions.

The current study showed a significant increase in metastatic and preoperative groups when compared to controls. A similar elevation of M2-PK was recorded by others [16, 17] in the metastatic breast cancer. Recently, studies documented a cross-talk between extracellular M2-PK and EGFR in TNBC cells for promotion of tumor growth, proliferation, and immune escape [58, 59]. Ectopic M2-PK also promotes stemness of breast cells via activation of Wnt/ $\beta$ -catenin signaling pathway and thus relates to poor prognosis (including relapse, metastasis, and drug resistance) in breast cancer patients [60].

In the present study, TuM2-PK level significantly related to menstruation status, and liver metastasis in preoperative group but no prior reports shown similar relations. Further studies are mandatory to validate this relation.

Our results showed a significant increase in both CRP and CA 15-3 in metastatic breast cancer when compared to the preoperative group. CA 15-3 also increased significantly in preoperative and metastatic groups when compared to controls. Inflammation and its components have been shown to affect tumor metabolism, growth, vascularity, invasion, and metastasis [61, 62, 63, 64, 65]. Ahmed et al. [66] reported a significant increase in CRP in metastatic breast cancer (Mean $\pm$ SD: 42.82 $\pm$ 4.23) than non-metastatic (Mean $\pm$ SD: 17.82 $\pm$ 1.27) and controls (Mean $\pm$ SD: 7.5 $\pm$ 0.11) among Egyptian females. They concluded that CRP can be used in the differential diagnosis for metastatic and non-metastatic breast cancer. DiGioia et al. [67] showed a significant difference between controls and metastatic breast cancer (MBC) ( $p=$ 0.003) at the baseline, and true positive and true negative patients at the time of metastasis ( $p=$ 0.000006). Although there was no statistical difference between controls and MBC in CA 15-3 at the baseline, there was a significant difference between true positive and true negative patients at the time of metastasis ( $p=$ 0.00006).

In the initial follow up period (40–50 months), the survival rate decreased significantly in our patients with TuM2-PK $>$ 15 U/ml in the metastatic group. No significant difference was observed between survival rate and TuM2-PK level in preoperative group. Far to our knowledge, this is the first study to compare the metastatic and preoperative breast cancer. In Melanoma, periampullary malignancy, and colorectal cancer, TuM2-PK with cut off values $\geqslant$ 15 U/ml ( $=$ 15 for melanoma, $>$ 27 for periampullary malignancy, and $>$ 20 for colorectal cancer) were suggested as predictors of survival [68, 69, 70].

TuM2-PK showed 59, 75.67% sensitivity (the proportion of patients with disease who test positive) and 73% specificity (is the proportion of patients without disease who test negative) (at the cut off value ( $\geqslant$ 15 U/ml) in our metastatic and preoperative groups; respectively. Quite similarly, Lüftner et al. [15] reported 85% specificity of TuM2-PK in advanced breast cancer. A sensitivity of 82% and a specificity of 60% was shown for TuM2-PK in squamous cell carcinoma of the cervix [57]. This indicates that TuM2-PK is not site-specific marker but is helpful to differentiate between malignant and non-malignant lesions.

CA 15-3 and TuM2-PK showed high diagnostic performance in metastatic and preoperative groups; respectively. Far to our knowledge, no similar results were reported before.

After 20 years follow up period, none of the studied markers shown any significant relation with patients’ prognosis in both metastatic or preoperative groups. From 50 months till 20 years, the death rate might be influenced by other morbidity factors.

In conclusion, this study correlates TuM2PK with tumor size, CRP and CA 15-3 in metastatic breast carcinomas. It documents the prognostic role of TuM2-PK and CA 15-3 in preoperative and metastatic breast cancer; respectively during the first 3–4 years follow up period. It further supports the relation between Tu-M2-PK level and survival in metastatic breast cancer during the first 3–4 years follow up period. For the first time, it negates the prognostic role of any of TuM2-PK, CRP, or CA 15-3 in breast cancer in long follow up period (20 years).

Conflict of interest

The authors declare that they have no competing interests.

References

Mazurek

et al., Pyruvate kinase type M2 and its role in tumor growth and spreading, Seminars in Cancer Biology 15(4) (2005), 300–308.

Christofk

H.R.

et al., The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumor growth, Nature 452(7184) (2008), 230–233.

Presek

Reinacher

and Eigenbrodt

, Pyruvate kinase type M2 is phosphorylated at tyrosine residues in cells transformed by Rouse sarcoma virus, FEBS Letters 242(1) (1988), 194–198.

Zwerschke

et al., Modulation of type M2 pyruvate kinase activity by the human papillomavirus type 16 E7 oncoprotein, Proceedings of the National Academy of Sciences of the United States of America 96(4) (1999), 1291–1296.

Zwerschke

and Jansen-Dürr

, Cell transformation by the E7 oncoprotein of human papillomavirus type 16: Interactions with nuclear and cytoplasmic target proteins, Advances in Cancer Research 78 (2001), 1–29.

Mazurek

et al., Metabolic cooperation between different oncogenes during cell transformation: Interaction between activated ras and HPV-16 E7, Oncogene 20 (47) (2001), 6891–6898.

Christofk

H.R.

et al., Pyruvate kinase M2 is a phospho- tyrosine-binding protein, Nature 452(7184) (2008), 181–186.

Mazurek

et al., Regulation of pyruvate kinase type M2 by A-Raf: A possible glycolytic stop or go mechanism, Anticancer Research 27(6B) (2007), 3963–3971.

Spoden

G.A.

et al., Isotype-specific inhibitors of the glycolytic key regulator pyruvate kinase subtype M2 moderately decelerate tumor cell proliferation, International Journal of Cancer 123(2) (2008), 312–321.

10.

Eigenbrodt

Mazurek

and Friis

R.R.

, Double role of pyruvate kinase type M2 in the regulation of phosphometabolite pools, In: Cell Growth and Oncogenesis Bannasch

Kanduc

Papa

and Tager

J.M.

, eds, 1

{}^{\rm st}

ed. Basel, Switzerland: Birkhauser Verlag, 1998, pp. 15–30.

11.

Eigenbrodt

Gerbracht

Mazurek

Presek

and Friis

, Carbohydrates metabolism and neoplasia: New perspectives for diagnosis and therapy, In: Biochemical and Molecular Aspects of Selected Cancers Pretlow

T.G.

Pretlow

T.P.

, eds, Volume II. Academic press, San Diego, 1994, pp. 311–381.

12.

Gatenby

R.A.

and Gillies

R.J.

, Why do cancers have high aerobic glycolysis? Nature Reviews Cancer 4(11) (2004), 891–899.

13.

DeBerardinis

R.J.

et al., The biology of cancer: Metabolic reprogramming fuels cell growth and proliferation, Cell Metabolism 7(1) (2008), 11–20.

14.

Mazurek

et al., Tumor M2-PK and glutaminolytic enzymes in the metabolic shift of tumor cells, Anticancer Research 20(6D) (2000), 5151–5154.

15.

Lüftner

et al., Tumor type M2 pyruvate kinase expression in advanced breast cancer, Anticancer Research 20(6D) (2000), 5077–5082.

16.

Lüftner

et al., Plasma levels of HER-2/neu, tumor type M2 pyruvate kinase and its tyrosine-phosphorylated metabolite in advanced breast cancer, Anticancer Research 23(2A) (2003), 991–997.

17.

Hoopmann

et al., Tumor M2 pyruvate kinase-determina- tion in breast cancer patients receiving trastuzumab therapy, Cancer Letters 187(1–2) (2002), 223–228.

18.

Coussens

L.M.

and Werb

, Inflammation and cancer, Nature 420 (2002), 860–867.

19.

Black

Kushner

and Samols

, C-reactive protein, The Journal of Biological Chemistry 279(47) (2004), 48487–48490.

20.

Gabay

and Kushner

, Acute-phase proteins and other systemic responses to inflammation, The New England Journal of Medicine 340(6) (1999), 448–454.

21.

Pepys

M.B.

and Hirschfield

G.M.

, C-reactive protein: A critical update, The Journal of Clinical Investigation 111(12) (2003), 1805–1812.

22.

Heikkilä

Ebrahim

and Lawlor

D.A.

, A systematic review of the association between circulating concentrations of C reactive protein and cancer, Journal of Epidemiology and Community Health 61(9) (2007), 824–833.

23.

Hanahan

and Weinberg

R.A.

, The hallmarks of cancer, Cell 100(1) (2000), 57–70.

24.

Asegaonkar

S.B.

et al., C-reactive protein and breast cancer: New insights from old molecule, International Journal of Breast Cancer 2015 (2015), 145647.

25.

Allin

K.H.

and Nordestgaard

B.G.

, Elevated C-reactive protein in the diagnosis, prognosis, and cause of cancer, Critical Reviews in Clinical Laboratory Sciences 48(4) (2011), 155–170.

26.

Gendler

S.J.

et al., Structure and biology of a carcinoma-associated mucin, MUC1, The American Review of Respiratory Disease 144(3 Pt 2) (1991), S42–47.

27.

Baldus

S.E.

Engelmann

and Hanisch

F.G.

, MUC1 and the MUCs: A family of human mucins with impact in cancer biology, Critical Reviews in Clinical Laboratory Sciences 41(2) (2004), 189–231.

28.

Duffy

M.J.

Evoy

and McDermott

E.W.

, CA 15-3: Uses and limitation as a biomarker for breast cancer, Clinica Chimica Acta 411(23–24) (2010), 1869–1874.

29.

Duffy

M.J.

et al., Biomarkers in breast cancer: Where are we and where are we going? Advances in Clinical Chemistry 71 (2015), 1–23.

30.

Mondal

H.P.

et al., Usefulness of serum CA-15.3 in the management of benign breast lesion, Journal of the Indian Medical Association 110(4) (2012), 242–244.

31.

Atoum

et al., Relationships among serum CA15-3 tumor marker, TNM staging, and estrogen and progesterone receptor expression in benign and malignant breast lesions, Asian Pacific Journal of Cancer Prevention 13(3) (2012), 857–860.

32.

Bombardieri

et al., CA 15.3 determination in patients with breast cancer: Clinical utility for the detection of distant metastases, European Journal of Cancer 29A(1) (1992), 144–146.

33.

Lee

J.S.

et al., Elevated levels of serum tumor markers CA 15-3 and CEA are prognostic factors for diagnosis of metastatic breast cancers, Breast Cancer Research and Treatment 141(3) (2013), 477–484.

34.

Harris

et al., American society of clinical oncology 2007 update of recommendations for the use of tumor markers in breast cancer, Journal of Clinical Oncology 25(33) (2007), 5287–5312.

35.

Shering

S.G.

et al., Preoperative CA 15-3 concentrations predict outcome of patients with breast carcinoma, Cancer 83(12) (1998), 2521–2527.

36.

Park

B.W.

et al., Preoperative CA 15-3 and CEA serum levels as predictor for breast cancer outcomes, Annals of Oncology 19(4) (2008), 675–681.

37.

Cañizares

et al., Preoperative values of CA 15-3 and CEA as prognostic factors in breast cancer: A multivariate analysis, Tumour Biology 22(5) (2001), 273–281.

38.

Gion

et al., Prognostic role of serum CA15.3 in 362 node-negative breast cancers, an old player for a new game, European Journal of Cancer 38(9) (2002), 1181–1188.

39.

Kumpulainen

E.J.

Keskikuru

R.J.

and Johansson

R.T.

, Serum tumor marker CA 15.3 and stage are the two most powerful predictors of survival in primary breast cancer, Breast Cancer Research and Treatment 76(2) (2002), 95–102.

40.

Molina

et al., Prospective evaluation of tumor markers (c-erbB-2 oncoprotein, CEA and CA 15.3) in patients with locoregional breast cancer, Anticancer Research 23(2A) (2003), 1043–1050.

41.

Duffy

M.J.

et al., High preoperative CA 15-3 concentrations predict adverse outcome in node-negative and node-positive breast cancer: Study of 600 patients with histologically confirmed breast cancer, Clinical Chemistry 50(3) (2004), 559–563.

42.

Martínez-Trufero

et al., Serum markers and prognosis in locally advanced breast cancer, Tumori 91(6) (2005), 522–530.

43.

McLaughlin

et al., The prognostic value of the tumor marker CA 15-3 at initial diagnosis of patients with breast cancer, The International Journal of Biological Markers 15(4) (2000), 340–342.

44.

The Belmont report: Ethical principles and guidelines for the protection of human subjects of research, The national commission for the protection of human subjects of biomedical and behavioral research. Retrieved at: (April 28, 2017) 1979. Available from: hhs.gov/ohrp/humansub-jects/guidance/belmont.html.

45.

El-Abd

et al., Clinical relevance of Hif-1α, Cox-2, leptin, and prolactin as hypoxic markers in breast cancer, Austral-Asian Journal Cancer 11(4) (2012), 237–246.

46.

Haybittle

J.L.

et al., A prognostic index in primary breast cancer, British Journal of Cancer 45(3) (1982), 361–366.

47.

Lee

A.H.

and Ellis

I.O.

, The Nottingham prognostic index for invasive carcinoma of the breast, Pathology Oncology Research 14(2) (2008), 113–115.

48.

Mehla

and Singh

P.K.

, MUC1: A novel metabolic master regulator, Biochimica Et Biophysica Acta 1845(2) (2014), 126–135.

49.

Viry

et al., Autophagy: An adaptive metabolic response to stress shaping the antitumor immunity, Biochemical Pharmacology 92(1) (2014), 31–42.

50.

Alves-Filho

J.C.

and Pålsson-McDermott

E.M.

, Pyruvate kinase M2: A potential target for regulating inflammation, Frontiers in Immunology 7 (2016), 145.

51.

Shirai

et al., The glycolytic enzyme PKM2 bridges metabolic and inflammatory dysfunction in coronary artery disease, The Journal of Experimental Medicine 213(3) (2016), 337–354.

52.

Spoden

G.A.

et al., Pyruvate kinase isoenzyme M2 is a glycolytic sensor differentially regulating cell proliferation, cell size and apoptotic cell death dependent on glucose supply, Experimental Cell Research 315(16) (2009), 2765–2774.

53.

Dong

et al., Altered glycometabolism affects both clinical features and prognosis of triple-negative and neoadjuvant chemotherapy-treated breast cancer, Tumour Biology 37(6) (2016), 8159–8168.

54.

Oremek

G.M.

et al., Pre-analytical problems in the measurement of tumor type pyruvate kinase (tumor M2-PK), Anticancer Research 23(2A) (2003), 1127–1130.

55.

Oehler

et al., Polytrauma induces increased expression of pyruvate kinase in neutrophils, Blood 95(3) (2000), 1086–1092.

56.

Landt

et al., Tumor-specific correlation of tumor M2 pyruvate kinase in pre-invasive, invasive and recurrent cervical cancer, Anticancer Research 30(2) (2010), 375–381.

57.

Kaura

Bagga

and Patel

F.D.

, Evaluation of the Pyruvate Kinase isoenzyme tumor (Tu M2-PK) as a tumor marker for cervical carcinoma, The Journal of Obstetrics and Gynaecology Research 30(3) (2004), 193–196.

58.

Lim

S.O.

et al., EGFR signaling enhances aerobic glycolysis in triple negative breast cancer cells to promote tumor growth and immune escape, Cancer Research 76(5) (2016), 1284–1296.

59.

Hsu

M.C.

et al., Extracellular PKM2 induces cancer proliferation by activating the EGFR signaling pathway, American Journal of Cancer Research 6(3) (2016), 628–638.

60.

Zhao

et al., PKM2 promotes stemness of breast cancer cell by through Wnt/β-catenin pathway, Tumour Biology 37(3) (2016), 4223–4234.

61.

Leek

R.D.

et al., Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma, Cancer Research 56(20) (1996), 4625–4629.

62.

Liao

et al., Cancer associated fibroblasts promote tumor growth and metastasis by modulating the tumor immune microenvironment in a 4T1 murine breast cancer model, PLoS One 4(11) (2009), e7965.

63.

Grivennikov

S.I.

Greten

F.R.

and Karin

, Immunity, inflammation, and cancer, Cell 140(6) (2010), 883–899.

64.

Ham

and Moon

, Inflammatory and microenvironmental factors involved in breast cancer progression, Archives of Pharmacal Research 36(12) (2013), 1419–1431.

65.

Guinan

E.M.

et al., The presentation of metabolic dysfunction and the relationship with energy output in breast cancer survivors: A cross-sectional study, Nutrition Journal 12 (2013), 99.

66.

Ahmed

S.A.

Hamed

M.A.

and Omar

O.S.

, Clinical utility of certain biomarkers as predictors of breast cancer with or without metastasis among Egyptian females, Tumour Biology 36(2) (2015), 815–822.

67.

Di Gioia

et al., Tumor markers in the early detection of tumor recurrence in breast cancer patients: CA 125, CYFRA 21-1, HER2 shed antigen, LDH and CRP in combination with CEA and CA 15-3, Clinica Chimica Acta 461 (2016), 1–7.

68.

Ugurel

et al., Tumor type M2 pyruvate kinase (TuM2-PK) as a novel plasma tumor marker in melanoma, International Journal of Cancer 117(5) (2005), 825–830.

69.

Goonetilleke

K.S.

et al., Diagnostic and prognostic value of plasma tumor M2 pyruvate kinase in periampullary cancer: Evidence for a novel biological marker of adverse prognosis, Pancreas 34(3) (2007), 318–324.

70.

Fatela-Cantillo

et al., Prognostic value of plasmatic tumor M2 pyruvate kinase and carcinoembryonic antigen in the survival of colorectal cancer patients, Tumour Biology 33(3) (2012), 825–832.

Plasma TuM2-PK correlates with tumor size,CRP and CA 15-3 in metastatic breast carcinomas;short versus long term follow up study of the Egyptian breast cancer patients

Abstract

BACKGROUND

AIM

PATIENTS AND METHODS

RESULTS

CONCLUSION

Keywords

1. Introduction

1.1 TuM2-PK

Table 1 Age, tumor prognostic factors, and patients ‘outcomes of the studied groups

1.3 Cancer antigen or carbohydrate antigen 15-3 (CA 15-3)

Table 2 TuM2-PK among the studied groups

3. Results

3.1 TuM2-PK

3.2 CA 15-3

3.3 CRP

3.4 Clinical outcome

4. Discussion

Conflict of interest

References

Table 1
Age, tumor prognostic factors, and patients ‘outcomes of the studied groups

Table 2
TuM2-PK among the studied groups