The Correlation Between Essential Amino Acid Tryptophan,Lysine,Phenylalanine and Chemotherapy of Breast Cancer

Abstract

To investigate the differences in serum tryptophan, lysine, and phenylalanine levels in breast cancer patients, the correlation between the three amino acids with the chemotherapy regimen, and their significance in the clinical diagnosis and treatment of breast cancer.

Clinical data were collected from the Department of Breast Surgery at Yunnan Cancer Hospital, encompassing 216 cases from July to December 2020, including 91 healthy individuals, 38 with benign tumors, and 87 with cancer. Amino acid levels were measured using liquid chromatography-tandem mass spectrometry. Statistical analyses, such as the Kruskal-Wallis H-test and Wilcoxon test, were conducted to compare the levels of these amino acids across the healthy group, benign tumor group, and breast cancer group. The χ2 test and Fisher's exact probability method were employed to assess the relationship between amino acid levels and breast cancer stage, grade, and chemotherapy regimen.

The results indicated that there were significant differences in serum lysine (H = 36.13, P < .001) and phenylalanine (H = 34.03, P < .001) levels among the three groups. However, tryptophan levels did not show statistically significant variances. Specifically, lysine and phenylalanine levels were significantly different when comparing the healthy group with the breast cancer group and the benign tumor group with the breast cancer group. These differences were not significant when comparing the healthy group with the benign tumor group. Furthermore, there were no statistically significant distinctions observed in lysine (F = 0.836, P > .05) and phenylalanine (F = 1.466, P > .05) levels across different conventional chemotherapy regimens among the breast cancer cases studied.

Serum lysine and phenylalanine levels might serve as potential biomarkers for breast cancer, and the choice of chemotherapy regimen is unlikely to impact significant changes in these amino acid levels.

Keywords

phenylalanine lysine tryptophan breast cancer chemotherapy

Introduction

Breast cancer has surpassed lung cancer as the leading cause of cancer incidence worldwide, with an estimated 2.3 million new cases in 2020, accounting for 11.7% of all cancer cases.¹ Chemotherapy is a systemic treatment that requires the selection of an optimal treatment regimen based on the breast cancer staging of different patients. Currently, treatment consists of the concurrent application of two or three combinations of the following carboplatin, cyclophosphamide, 5-fluorouracil/capecitabine, paclitaxel (paclitaxel, docetaxel), and anthracycline (adriamycin, epothilone)² The following is a list of the most popular drugs used in the treatment of cancer.

Amino acids are indispensable for the growth of cancer cells, both essential and nonessential, and serve as important signaling molecules that regulate metabolic pathways, protein translation, autophagy, defense against reactive oxygen species, and many other functions.³ Studies over the past decades have demonstrated the important role of amino acids in cancer metabolism, where amino acids are involved in providing energy and raw materials for cancer cell growth, enhancing tumor invasiveness by affecting redox homeostasis and epigenetic regulation, and certain metabolic intermediates can promote tumorigenic and antitumor activity.⁴ The results of Dan et al confirm that the metabolism of amino acids is accelerated in breast tumor cells, that a significant decrease in amino acid concentration between the breast cancer group and the control group is positively correlated with the progression of breast cancer, and that the five free amino acids (arginine, alanine, isoleucine, tyrosine, and tryptophan) may serve as good potential discriminators between breast cancer patients and healthy patients in future studies.⁵

Amino acids have been associated with the development of a variety of diseases, and tryptophan metabolism is involved in intestinal homeostasis⁶ and cardiovascular system regulation^7,8 Lysine stimulates gastrointestinal activity. Lysine stimulates gastrointestinal activity⁹ and in a lipopolysaccharide-induced mouse model, lysine significantly reduced the inflammatory response to acute lung injury¹⁰ Lysine significantly reduced the inflammatory response to acute lung injury in a lipopolysaccharide-induced mouse model. Phenylalanine levels predicted the development of diabetes mellitus and, by increasing the propensity for atherosclerosis, may predict cardiovascular events during long-term follow-up¹¹ Meanwhile, phenylalanine induced a mouse model of pulmonary hypertension¹² and its levels were independently and positively correlated with disease severity in COVID-19 patients¹³ and tryptophan concentrations significantly decreased with disease severity.¹⁴

Studies have found that changes in amino acids are specific to certain types of cancer.¹⁵ A study by Gu et al found that: histidine levels were significantly decreased in breast cancer patients. Serum serine, alanine, valine, lysine, and histidine levels were significantly lower in gastric cancer patients. While methionine, leucine, tyrosine and lysine levels were significantly higher in thyroid cancer patients. Studies have also shown that alanine, arginine, aspartic acid, and cysteine promote breast cancer cell proliferation. Although the relationship between amino acids and certain types of cancer was discovered early on, only a few studies have investigated the use of amino acids in diagnosis and treatment.¹⁶

It has been shown that tryptophan metabolism and its products inhibit the ability of effector T cells to suppress anti-tumor immunity and the proliferation of natural killer cells, reduce the immunogenicity of dendritic cells, and promote the formation of a tumor immune microenvironment,¹⁷ can strongly promote cancer cell migration and invasion, and tryptophan production of nicotinamide adenine dinucleotide (NAD +) confers resistance to oxidative stress induced by radiochemotherapy.¹⁸. Interference with lysine methylation of transcription factors in vivo and in vitro inhibits cancer cell proliferation and reverses tumor progression.¹⁹ Lysine deficiency may lead to immunodeficiency and promote cancer cell proliferation.²⁰ In triple-negative breast cancer, lysine depletion inhibits cancer cell proliferation and decreases cell viability.²¹ Phenylalanine levels are valuable in identifying patients with metastatic or limited breast cancer.²² Phenylalanine metabolism is involved in regulating the suppression of T cell immune responses.²³

Different amino acid levels have different levels of impact on tumors, and tumor treatments may alter amino acid metabolism, thereby affecting the tumor microenvironment and influencing response to treatment.¹ Do amino acid levels change after chemotherapy, and do changes in amino acid levels differ after treatment with different chemotherapy regimens?

In order to explore the above questions, we intend to retrospectively study the changes in the levels of three amino acids in breast cancer patients and their differences in different chemotherapy regimens for breast cancer. To explore the correlation between the differences in tryptophan, lysine and phenylalanine levels in breast cancer patients with different chemotherapy regimens and their significance for clinical diagnosis and treatment.

Information and Methodology

General Information

The clinical data of 87 breast cancer patients (BC group) admitted to the Department of Breast Surgery I of Yunnan Cancer Hospital from July 2020 to December 2020 were selected for retrospective analysis, and the patients were all female, aged 23-76 years old, with an average age of 49 ± 9 years old. Diagnostic criteria of breast cancer group: patients were diagnosed by more than two pathologists and met the clinical diagnostic criteria of breast cancer. Inclusion criteria of breast cancer group: patients were diagnosed with breast cancer by pathological examination and clinical diagnosis. Exclusion criteria: ①complicated with other malignant tumors; ②combined with other major diseases; ③with contraindications to chemotherapy or those who cannot tolerate chemotherapy; ④lactating or pregnant women. The molecular typing and TNM staging of breast cancer were based on the “2021 Chinese Anti-Cancer Association Breast Cancer Diagnostic and Treatment Guidelines and Norms”. There were 33 cases of Luminal-A, 21 cases of Luminal-B, 12 cases of Her-2 positive, 10 cases of triple-negative and 3 cases of unknown, 26 cases of Stage-I, 32 cases of Stage-II, 20 cases of Stage-III, 3 cases of Stage-IV and 6 cases of others. There were 64 cases of estrogen receptor positivity, 72 cases of progesterone receptor positivity, and 23 cases of human epidermal growth factor receptor positivity (Figure 1). Another 91 healthy women and 38 women with benign breast tumors who were examined at the same time in our department were selected as the control group. Inclusion criteria for the healthy group (HD): women were physically healthy, without major diseases, and had not taken any medication 1 week before blood collection; inclusion criteria for the benign breast tumor group (BE): benign lesions were shown by breast tissue puncture or pathology examination. Clinical data of the breast cancer study subjects were collected, including gender, age, body mass, height, chemotherapy regimen, pathologic type, clinical stage and histologic typing status. The study was agreed and signed by the study subjects with informed consent, and approved by the Ethics Committee of Yunnan Cancer Hospital.

Figure 1.

Patient selection process for breast cancer group.

Laboratory Methods

All 61 cases of chemotherapy patients in the breast cancer group collected peripheral venous blood by fasting before 8:00 on the second day after the completion of the first chemotherapy cycle, the rest of the breast cancer patients and benign breast tumors group collected peripheral venous blood by fasting at 8:00 on the second day after the diagnosis, and the patients in the healthy group collected peripheral venous blood by fasting on the day of physical examination. Each study subject had 3-4 mL of blood taken from each, placed in procoagulant tubes, and immediately sent to Yunnan Cancer Hospital Research Institute for serum amino acid level testing using liquid chromatography-tandem mass spectrometry . The normal range for tryptophan levels was 29-77 μmol/L, for phenylalanine levels was 35-120 μmol/L, and for lysine levels was 119-300 μmol/L. The serum levels of tryptophan, lysine, and phenylalanine were statistically compared between HD, BE, and BC.

Statistical Methods

SPSS 22.0 and GraphPad Prism 9.0 software were used for statistical analysis and plotting. The levels of tryptophan, lysine and phenylalanine in the healthy control group, the benign breast tumor group and the breast cancer group were tested by the K-S (Kolmogorov-Smirov) test and the P-P plot test to show that they were not normally distributed, the amino acid levels were expressed as the median and interquartile spacing, and analyzed by the Kruskal-Wallis H test. Intergroup analysis was performed using the wilcoxon test. Clinical baseline data of patients in the breast cancer group, TNM staging of different breast cancers, and pathological staging were analyzed using the χ2 test or Fisher's exact probability method, and a two-sided P value <.05 was considered statistically significant.

Result

Levels of Amino Acids in Different Groups and Their inter-Group Differences

The differences in the levels of tryptophan, lysine, and phenylalanine were analyzed among HD, BE, and BC. The results showed that there was no statistically significant difference in tryptophan levels among the healthy control group (60.55 ± 16.11), the benign breast tumor group (60.14 ± 22.35), and the breast cancer group (62.11 ± 21.96) (H = 1.51 and P > .05). There was a statistically significant difference in lysine levels among the HD (134.04 ± 52.08), BE (146.48 ± 47.06), and BC (181.84 ± 68.17) (H = 36.13, P < .001). Similarly, the difference in phenylalanine levels was statistically significant (H = 34.03, P < .001) in the comparison of healthy control group (52.23 ± 11.43), benign breast tumor group (51.39 ± 10.97) and breast cancer group (67.47 ± 30.12). Lysine levels and phenylalanine levels were higher in the breast cancer group compared to the healthy control and benign breast tumor groups. (Table 1)

Table 1.

Levels of Amino Acids in Different Groups and Their inter-Group Differences (μmol/L).

	HD（M±IΘP）	BE（M±IΘP）	BC （` M±IΘP）	H	P
Tryptophan	60.55 ± 16.11	60.14 ± 22.35	62.11 ± 21.96	1.51	.47
Lysine	134.04 ± 52.08	146.48 ± 47.06	181.84 ± 68.17	36.13	＜.001*
Phenylalanine	52.23 ± 11.43	51.39 ± 10.97	67.47 ± 30.12	34.03	＜.001*

*P ＜ .05.

Among the three groups of subjects, phenylalanine and lysine with statistically significant differences were further analyzed. In the 91 cases of the healthy control group, the levels of phenylalanine in all subjects were within the normal range, while 27 cases had lysine levels below the normal range. For the 38 cases in the benign breast cancer group, the levels of phenylalanine in all subjects were within the normal range, with 8 cases having slightly low lysine levels. In the breast cancer group of 87 cases, 2 cases had low phenylalanine levels, 3 cases had lysine levels above the normal range, and 12 cases had low lysine levels.(Figure 2)

Figure 2.

The level of phenylalanine and lysine in the three groups of subjects.

Comparison of Levels of Tryptophan, Lysine, and Phenylalanine Between HD Versus BE, HD Versus BC, and BE Versus BC

The levels of tryptophan, lysine, and phenylalanine were compared between HD &BE, HD &BC, and BE & BC. The results showed that there were no statistically significant differences in the levels of tryptophan between HD versus BE, HD versus BC, BE versus BC (P > .05). Lysine levels showed statistically significant differences in comparisons between HD versus BC (P < .05), and BE versus BC (P < .05), while no statistically significant differences were observed in the comparison between HD versus BE (P > .05). Phenylalanine levels showed statistically significant differences in comparisons between HD versus BC (P < .05), and BE versus BC (P < .05), while no statistically significant differences were observed in HD versus BE (P > .05). (Table 2, Figure 3)

Figure 3.

Compares the levels of tryptophan, lysine, and phenylalanine in the healthy group versus the benign breast tumor group, the healthy group versus the breast cancer group, and the breast cancer group versus the benign breast tumor group. Panel A: Comparison of tryptophan levels in the healthy group versus the benign breast tumor group, the healthy group versus the breast cancer group, and the breast cancer group versus the benign breast tumor group; Panel B: Comparison of lysine and tryptophan levels in the healthy group versus the benign breast tumor group, the healthy group versus the breast cancer group, and the breast cancer group versus the benign breast tumor group; Panel C: Comparison of phenylalanine levels in the healthy group versus the benign breast tumor group, the healthy group versus the breast cancer group, and the breast cancer group versus the benign breast tumor group. (*** indicates P ＜ .001, HD: healthy group; BE: benign breast tumor group; BC: breast cancer group).

Table 2.

Comparison of Levels of Tryptophan, Lysine, and Phenylalanine Between the Healthy Group and the Benign Breast Tumor Group, the Healthy Group and the Breast Cancer Group, and the Benign Breast Tumor Group and the Breast Cancer Group.

Group	Tryptophan	Lysine	Lysine
HD versus BE	0.558	0.135	0.965
HD versus BC	0.395	＜0.001	＜0.001
BE versus BC	0.269	＜0.001	＜0.001

HD : healthy group ; BE : benign breast tumor group ; BC:breast cancer group

Comparison of Different Amino Acid Levels with the Baseline Clinical Characteristics of Breast Cancer Patients

The above analysis results show that the differences in lysine and phenylalanine levels among HD, BE, and BC are statistically significant (P ＜ .05), while the difference in tryptophan level is not statistically significant (P ＞ .05). To further investigate these differences, a baseline clinical analysis was conducted on 87 breast cancer patients. Based on the median levels of lysine and phenylalanine in these 87 breast cancer patients, they were divided into four subgroups. Among them, 43 cases had lysine levels <181.84 μmol/L, and 44 cases had lysine levels ≥181.84 μmol/L. The comparison between the two groups in terms of menopausal status showed statistical differences (χ2 = 14.345, P ＜ .05), while no statistically significant differences were found in age, BMI, TNM staging, tumor size, presence of lymph node metastasis, ER, PR, Her-2, Ki-67, and pathological subtypes (P ＞ .05). Similarly, 43 cases had phenylalanine levels <67.47 μmol·L-1, and 44 cases had phenylalanine levels ≥67.47 μmol·L-1. The comparison between the two groups in terms of menopausal status showed statistical differences (χ2 = 5.484, P ＜ .05), while no statistically significant differences were found in age, BMI, TNM staging, tumor size, presence of lymph node metastasis, ER, PR, Her-2, Ki-67, and pathological subtypes (P ＞ .05). (Table 3)

Table 3.

The Clinical Baseline Characteristics of Breast Cancer Patients at Different Amino Acid Levels (μmol/L).

	Lysine				Phenylalanine
Characteristic	＜181.84（n = 43）	≥181.84（n = 44）	χ2	P-value	＜67.47 （n = 43）	≥67.47（n = 44）	χ2	P-value
Age(year)			1.946	.163			3.327	.068
＜50	18	25			17	26
≥50	25	19			26	18
Menopause			14.345	＜.001			5.484	.019
yes	20	4			17	7
no	23	38			26	35
unknown	0	2			0	2
BMI(kg/m²)			0.341	.559			1.008	.315
＜25	33	36			36	33
≥25	10	8			7	11
TNM Stage			0.521	.771			0.590	.745
I-II	29	29			30	28
III	9	11			9	11
IV	2	1			2	1
unknown	3	3			2	4
Tumor Size（l/cm)			4.989	.173			3.714	.294
Tx	4	4			4	4
＜2	10	15			9	16
2-5	28	20			27	18
＞5	1	5			3	3
Lymphatic metastasis			0.037	.848			＜0.001	＞.999
+	23	25			24	24
-	19	19			19	19
unknown	1	0			0	1
ER			2.591	.107			2.199	.138
+	28	36			29	35
-	14	8			14	8
unknown	1	0			0	1
PR			1.597	.206			0.341	.559
+	33	39			35	37
-	9	5			8	6
unknown	1	0			0	1
Her-2			0.102	.749			0.001	.975
+	11	12			12	11
-	29	27			29	27
unknown	3	5			2	6
Ki-67			0.752	.386			0.752	.386
＜14%	11	15			11	15
≥14%	32	29			32	29
Pathological type			4.919	.085			2.537	.281
ILC	0	3			2	1
IDC	35	39			35	39
DCIS	2	0			2	0
others	6	2			4	4

Abbreviations: ILC, invasive lobular carcinoma; IDC, invasive ductal carcinoma; DCIS, ductal carcinoma in situ.

Comparison of Amino Acid Levels in Breast Cancer Patients at Different TNM Stages

According to the 2021 edition of the Chinese Anti-Cancer Association's Breast Cancer Diagnosis and Treatment Guidelines, breast cancer was staged using TNM criteria. After excluding six cases with missing data, the remaining 81 patients were staged, with 26 cases at stage I, 32 cases at stage II, 20 cases at stage III, and 3 cases at stage IV. The results showed that there were no statistically significant differences in phenylalanine levels between stages I and II (P ＞ .05) or between stages III and IV (P ＞ .05). Similarly, there were no statistically significant differences in lysine levels between stages I and II (P ＞ .05) or between stages III and IV (P ＞ .05). (Table 4)

Table 4.

Comparison of Amino Acid Levels in Breast Cancer Patients at Different TNM Stages (μmol/L).

	Phenylalanine				Lysine
Stage	＜67.47	≥67.47	χ2	P	＜181.84	≥181.84	χ2	P
	n = 43	n = 44			n = 43	n = 44
			0.085	.771			0.279	.597
I	58.19 ± 17.56（n = 14）	78.04 ± 20.24（n = 12）			137.58 ± 66.43（n = 12）	138.97 ± 37.58（n = 14）
II	58.83 ± 15.19（n = 16）	82.66 ± 27.06（n = 16）			156.62 ± 43.75（n = 17）	216.72 ± 65.10（n = 15）
			0.491	.484			0.491	.484
III	46.91 ± 10.98（n = 9）	84.38 ± 18.16（n = 11）			148.02 ± 53.92（n = 96）	209.49 ± 54.74（n = 11）
IV	49.61（n = 2）	85.58（n = 1）			142.87（n = 2）	190.39（n = 1）
unknown	2	4			3	3

Comparison of Amino Acid Levels in Breast Cancer Patients with Different Molecular Subtypes

According to the 2021 edition of the Chinese Anti-Cancer Association's Breast Cancer Diagnosis and Treatment Guidelines, patients were categorized into different molecular subtypes. After excluding 8 cases with missing data, the remaining 79 breast cancer patients were divided into four subtypes: including 33 cases of Luminal-A; 21 cases of Luminal-B: 12 cases of Her-2 positivity; 10 cases of triple-negativity and 3 cases of unknown. The results showed that there was no statistically significant difference in lysine levels in the comparison of different molecular typing (χ 2 = 1.237, P > .05). Phenylalanine levels were not statistically significant in the comparison of different molecular typing (χ 2 = 1.581, P > .05). (Table 5)

Table 5.

Amino Acid Levels in Breast Cancer Patients with Different Molecular Subtypes (μmol/L).

Variables	Lysine				Phenylalanine
	＜181.84	≥181.84	χ2	P	＜67.47	≥67.47	χ2	P
	n = 39	n = 40			n = 37	n = 42
Molecular type			1.237	.744			1.581	.664
Luminal-A	142.00 ± 58.64（n = 14）	209.49 ± 61.64（n = 19）			50.09 ± 23.60（n = 15）	74.13 ± 34.52（n = 18）
Luminal-B	167.39 ± 24.72（n = 11）	225.26 ± 73.76（n = 10）			58.56 ± 9.62（n = 8）	84.90 ± 10.12（n = 13）
Her-2(+)	127.51 ± 9.99（n = 7）	206.37 ± 28.19（n = 5）			48.47 ± 21.40（n = 7）	84.38 ± 26.53（n = 5）
Triple-negative	117.66 ± 100.58（n = 5）	245.05 ± 65.44（n = 5）			44.41 ± 36.19（n = 5）	82.68 ± 21.47（n = 5）
Unknown	n = 2	n = 1			n = 2	n = 1

Comparison of Amino Acid Levels in Breast Cancer Patients Undergoing Different Chemotherapy Regimens

Excluding patients with missing data, amino acid levels were compared among the remaining 61 breast cancer patients. Among them, 11 cases received the TC regimen (paclitaxel and cyclophosphamide), 22 cases received the AC regimen (doxorubicin/cyclophosphamide), 22 cases received the AC/EC-T regimen (sequential doxorubicin/cyclophosphamide followed by paclitaxel), and 6 received other regimens. The results showed that there was no statistically significant difference in lysine levels among the three chemotherapy regimens (F = 0.836, P > .05). Similarly, there was no statistically significant difference in phenylalanine levels among the three chemotherapy regimens (F = 1.466, P > .05). (Table 6)

Table 6.

Comparison of Amino Acid Levels in Breast Cancer Patients Undergoing Different Chemotherapy Regimens (n = 61)[`c±s, (μmol/L)].

Variables	TC (n = 11）	AC (n = 22）	AC/EC-T (n = 22）	other (n = 6）	F	P
Lysine	184.38 ± 66.77	205.83 ± 61.78	200.44 ± 56.83	167.15 ± 50.80	.836	.480
Phenylalanine	65.55 ± 21.50	76.41 ± 21.06	73.66 ± 18.99	59.34 ± 25.41	1.466	0.233

Comparison of Chemotherapy Regimens in Breast Cancer Patients with Different Amino Acid Levels

The 61 breast cancer patients were grouped based on their lysine levels: 24 cases with lysine <181.84 μmol/L and 37 cases with lysine ≥181.84 μmol/L. The difference in lysine levels among the three chemotherapy regimens was not statistically significant (χ2 = 1.656, P > .05). Similarly, the phenylalanine levels were compared: 26 cases with phenylalanine <67.47 μmol/L and 35 cases with phenylalanine ≥67.47 μmol/L. The difference in phenylalanine levels among the three chemotherapy regimens was also not statistically significant (χ2 = 1.591, P > .05). (Table 7, Figure 4)

Figure 4.

A: Comparison of serum lysine and phenylalanine levels in breast cancer patients receiving the TC regimen; B: Comparison of serum lysine and phenylalanine levels in breast cancer patients receiving the AC regimen;C: comparison of serum lysine and phenylalanine levels in breast cancer patients receiving the AC/EC-T regimen.

Table 7.

Chemotherapy Regimens for Breast Cancer Patients with Different serum Amino Acid Levels(μmol/L).

Variables	Lysine				Phenylalanine
	＜181.84	≥181.84	χ2	P	＜67.47	≥67.47	χ2	P
	n = 24	n = 37			n = 26	n = 35
Chemotherapy regimen			1.656	.437			1.591	.451
TC	131.19 ± 50.94 (n = 6）	215.98 ± 77.34 (n = 5）			47.19 ± 12.80 (n = 6）	76.36 ± 28.48 (n = 5）
AC	171.80 ± 69.11 (n = 8）	232.28 ± 59.84 (n = 14）			57.17 ± 19.72 (n = 7）	85.58 ± 31.54 (n = 15）
AC/EC-T	140.68 ± 72.16 (n = 7）	212.17 ± 63.34 (n = 15）			59.26 ± 17.70 (n = 9）	84.23 ± 11.63 (n = 13）
other	(n = 3）	(n = 3）			(n = 4）	(n = 2）

Discussion

Breast cancer has become the most common cancer among women. Amino acid metabolism plays a crucial role in the progression of breast cancer.^4,5,24 Previous studies have reported the significant roles of glutamine,²⁵ arginine, cysteine, and tryptophan¹⁷ in the development of breast cancer.^4,23,26 However, there is limited research on the correlation between amino acid levels and breast cancer chemotherapy regimens. In this study, we investigated for the first time the levels of tryptophan, lysine, and phenylalanine in breast cancer, and their correlation with different chemotherapy regimens. We compared the differences in amino acid levels among breast cancer patients undergoing different chemotherapy regimens and discussed their implications for clinical diagnosis and treatment.

We found that there were differences in the levels of lysine and phenylalanine among the healthy group, benign breast tumor group, and breast cancer group. In the breast cancer group, the levels of lysine and phenylalanine were significantly higher than those in the healthy group and benign breast tumor group, while there was no difference in tryptophan levels. However, previous studies have indicated a decrease in tryptophan levels in breast cancer. Considering that amino acid levels are influenced by diet²⁷ and not excluding the possibility of amino acids adapting to the metabolic status of tumors,²⁸ our multiple comparisons revealed differences in the expression of lysine and phenylalanine levels between the breast cancer group and the healthy group. Our results also showed that there were differences in the expression of lysine and phenylalanine levels between the breast cancer group and the benign breast tumor group. Previous studies rarely included the benign breast tumor group as a control group. Oakman et al's study found that compared to early-stage breast cancer, metastatic breast cancer showed elevated levels of lysine and phenylalanine.²⁹ The above results indicate an increase in the expression of lysine and phenylalanine in breast cancer, and serum levels of lysine and phenylalanine may serve as potential biomarkers for breast cancer.

The levels of serum tryptophan, lysine, and phenylalanine in breast cancer are still inconclusive.³⁰ Most current studies indicate that compared to healthy groups, untreated primary breast cancer shows lower levels of tryptophan, phenylalanine, and lysine,^30,31 which differs from our research. However, in this study, the majority of collected serum amino acids are from treated breast cancer patients, considering the inhibition of amino acid metabolism in breast cancer after chemotherapy. The specific mechanism requires further investigation. In order to control for confounding factors, baseline levels were compared in 87 breast cancer patients, and the results showed that, except for differences in menopausal status, there were no significant differences in patient age, BMI, stage, tumor size, presence of lymph node metastasis, ER, PR, Her-2, Ki-67, and pathological type, indicating comparability between the groups.The study of 30 cases of breast ductal carcinoma by Dan et al showed that most amino acid levels increased from stage I to stage II, and then decreased from stage II to stage III.⁵ However, our analysis found no difference in the expression of lysine and phenylalanine levels between stages I and II, and stages III and IV in the breast cancer group (81 cases). Compared with the study of ductal carcinoma, we did not restrict the pathologic types of breast cancer, and whether there is a difference in amino acid metabolism among different pathologic types needs to be further investigated by enlarging the sample size. luminal typing comparison results: there was no difference in the expression of lysine and phenylalanine levels among different molecular typing and there was no significant difference. In this experiment, breast cancer typing had no effect on the expression of lysine and phenylalanine levels, which is consistent with previous studies.²² This is consistent with previous studies. Previous studies have shown that amino acid levels are affected by the age of the patient, surgery and changes in physical status, as well as gastrointestinal side effects caused by chemotherapy, which affect the gastrointestinal absorption of amino acids and then change the serum amino acid levels.²⁵ The effect of the amino acid absorption in the gastrointestinal tract will change the serum amino acid level.²²

Chemotherapy remains one of the main treatment methods for breast cancer, and chemotherapeutic drugs have varying degrees of impact on amino acid metabolism.³² In breast cancer, the expression of L-amino acid transporter (LAT1) for tryptophan and phenylalanine increases, and the level of LAT1 expression is correlated with the poor clinical prognosis of cancer patients. Tryptophan metabolism product indoleamine-2,3-dioxygenase (IDO) promotes tumor-induced immune system tolerance and suppression, and is associated with poor prognosis in chemotherapy. In paclitaxel-resistant breast cancer cells, the expression and activity of IDO significantly increase.³³ Lysine restriction can affect tumor cell growth,²¹ and the concentration of phenylalanine can predict the severity of future peripheral neuropathy caused by paclitaxel.

Chemotherapeutic drugs can influence the serum amino acid levels of breast cancer patients.^34–37 However, there are few reports on the correlation between lysine and phenylalanine levels and chemotherapy. In this study, under three common chemotherapy regimens, there was no significant difference in lysine and phenylalanine levels between different chemotherapy regimens. When comparing lysine <181.84 μmol/L with lysine ≥181.84 μmol/L, and phenylalanine <67.47 μmol/L with phenylalanine ≥67.47 μmol/L, there was no significant difference observed among the three chemotherapy regimens. Phenylalanine and lysine levels in breast cancer patients were not affected by chemotherapy regimens, and their levels did not affect the efficacy of chemotherapy.

However, we recognize that the small number of people in the different subgroups of phenylalanine and lysine in the different chemotherapy regimens may have an impact on the statistical results, and further expansion of the sample size is needed for an in-depth study.

In summary, phenylalanine and lysine levels exhibit differential expression between the breast cancer group, the healthy group, and the benign breast tumor group. Furthermore, the levels of phenylalanine and lysine in the healthy group are lower than those in the breast cancer group. Therefore, serum phenylalanine and lysine levels could serve as potential biomarkers for breast cancer. The levels of lysine and phenylalanine show no significant difference among different stages, molecular subtypes, and chemotherapy regimens. The levels of phenylalanine and lysine in breast cancer patients were not affected by chemotherapy regimens, and the effects of chemotherapeutic agents on the amino acid levels of breast cancer patients need to be analyzed in further experiments.

Conclusion

Phenylalanine and lysine levels were differentially expressed between the breast cancer group and the healthy and benign breast tumor groups, and serum phenylalanine and lysine levels can be used as potential biomarkers for breast cancer. Lysine and phenylalanine levels did not differ in expression among different stages, molecular typing and chemotherapy regimens, and the phenylalanine and lysine levels of breast cancer patients were not affected by chemotherapy regimens, and the choice of chemotherapy regimen did not affect the significant changes in lysine and phenylalanine levels. Due to the limited sample size of each molecular staging and chemotherapeutic regimen, further studies with larger data are needed.

Footnotes

Acknowledgements

We are grateful to all of the reviewers for their comments.

Availability of Data and Materials

The datasets used during the current study are available from the corresponding author on reasonable request.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethics Approval and Consent to Participate

The author is responsible for all aspects of the work to ensure that issues related to the accuracy and completeness of any part of the work are properly investigated and resolved. Subjects have given their written informed consent and that the study protocol was approved by the institute ‘s committee on human research. Study approval statement: This study complies with the Declaration of Helsinki (revised in 2013) and was approved by the ethics committee of the Third Affiliated Hospital of Kunming Medical University (Yunnan Cancer Hospital)(No.KY201944). Written informed consent was obtained from participants to participate in the study.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the cience and Technology Project of Yunnan Provincial Science and Technology Department, Wu Jieping Medical Foundation, National Natural Science Foundation of China, Beijing Science And Technology Innovation Medical Development Foundation, Yunnan Health Training Project of High Level Talents, (grant number no.202001AU070053, 202001AU070093 and 202201AY0700, no.320.6750.2022-19-58, no. 81960542, 82360614 and 81960517, no. KC2021-JK-0044-6, no.H-2019075).

ORCID iD

Shicong Tang

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