Occupational pesticide exposure is linked to the prevalence of Luminal B breast cancer and poor prognosis features in Brazilian rural women

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Occupational pesticide exposure is linked to the prevalence of Luminal B breast cancer and poor prognosis features in Brazilian rural women

https://doi.org/10.21203/rs.3.rs-4182249/v1

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Brazil is among the top three global consumers of pesticides, despite evidence concerning the negative impact on public health. The exposure on female rural workers has been neglected, and the incidence of pesticide-induced diseases such as breast cancer is growing. In this context, this study analyzed the impact of occupational/household pesticide exposure on the clinicopathological profile of breast cancer in rural women. Clinicopathological data were collected from medical records and analyzed from a total of 386 patients (208 exposed and 141 unexposed to pesticides). Was used descriptive and inferential statistics methods to characterize the patient data, including the chi-square test and Fisher's exact test, to evaluate associations between variables. This data was grouped as patient, tumor, and disease characteristics. Exposed patients had a prevalence of Luminal B subtype (32.83%), while unexposed patients had a prevalence of Luminal A molecular subtype (37.78%). Exposed patients also had higher disease recurrence (10.19%), chemoresistance (21.26%), and death occurrence (7.21%) than unexposed patients. Breast cancer patients exposed to pesticides were also more likely to have distant metastases and lymph node invasion compared to breast cancer unexposed patients. These findings indicate that pesticide exposure favors the occurrence of more aggressive breast cancer in rural women through occupational exposure. This results indicate that this occupational information could be added to the screening process and risk determination for breast cancer severity.

Biological sciences/Ecology/Agri ecology

Health sciences/Oncology

pesticides

breast cancer

luminal B subtype

chemoresistance

recurrence

The use of pesticides in agriculture began with the “Green Revolution” movement in the 50s, using the argument of increasing food production.¹ Unfortunately, at that time, there was no understanding of the potential risks of its use for the environment and human health.²

Several studies have demonstrated pesticide exposure risks to human health, such as their carcinogenic potential,^3,4 endocrine disruption,⁵ genotoxicity,^6–8 and compromised immune system.^9–12 With increased negative evidence on pesticide use, one of the main discussions in this field is the fact that several pesticides have carcinogenic potential. They have been classified by the International Agency for Cancer Research (IARC) as potentially, probably, or proven carcinogens. A variety of cancers have been linked to pesticide exposure, including thyroid, ¹³ colorectal,¹⁴ bladder,¹⁵ blood,¹⁶ brain,¹⁷ and breast cancer.¹⁸

In the context of pesticide health risks, it is important to note the growth of the feminization movement in agriculture; it has been estimated that women represent 43% of the world's agricultural workforce.¹⁹ This trend has been observed in several regions of the world, such as the European Union, where women represent 29% of rural workers,²⁰ Brazil, where they represent 45% of women, and certain regions of Africa and Asia, where women’s representation can reach up to 60%.²¹ The feminization of agriculture may lead to an increase in the incidence of breast cancer in women.

Due to the extensive exposure of rural women in agriculture and the endocrine disruption properties of pesticides, attention has been drawn to the incidence of female tumors, such as breast cancer. Several studies have addressed the relationship between pesticide exposure and the increased risk of developing this pathology.^22–24 Endocrine deregulation can occur by mainly two exposure models: programming, which modifies tissues during embryonic development until puberty, making them susceptible to cancer, and worsening, where subsequent exposure leads to malignant evolution of precancerous cells or benign lesions.²⁵

Considering little is known about the impact of chronic and continued occupational pesticide exposure on the clinicopathological profiling of breast cancer, the present study focused on understanding the issue. To reach this goal, we performed extensive data collection from patients diagnosed with breast cancer who had visited a public hospital located in Paraná state, a Brazilian setting known for its high use of pesticides and female rural work. Information concerning patient profiles and tumor characteristics was obtained and analyzed using descriptive and inferential statistical methods, aiming to determine a clinicopathological signature associated with pesticide exposure in women with breast cancer.

2.1. Study design and data collection

This is a descriptive, cross-sectional, and quantitative exploratory study with the objective of determining a clinicopathological signature associated with exposure to pesticides in rural women with breast cancer. Figure 1 illustrates the study design.

This study complied with the national and international regulatory standards for research involving humans and was approved by the Research Ethics Committee (CEP) of the State University of Western Paraná (UNIOESTE), under the number CAAE 35524814.4.0000.0107. All volunteers signed a free and informed consent form. Data, including unique materials, documentation, and codes used in the analysis, are available at https://github.com/Laboratorio-de-Analise-de-Dados/Article_pesticide_exposure.

A total of 923 women who attended the Francisco Beltrão Cancer Hospital (Ceonc) from May 2015 to April 2023 with images suggestive of breast lesions identified by mammograms and ultrasound were included. The study comprised the Eighth Paraná Health Region, which comprises 27 municipalities characterized predominantly by rural family work. To obtain the diagnosis of breast cancer, a biopsy of the suspicious lesion was performed by a pathologist, followed by anatomopathological analysis and immunohistochemistry. After excluding patients with benign lesions, 386 patients were included in the study as having a breast cancer diagnosis. Patients were categorized as occupationally exposed (n = 208) or unexposed to pesticides (n = 141).

The Eighth Regional Health Region of Paraná comprises about 500,000 inhabitants living in the following 27 municipalities: Ampére, Barracão, Bela Vista da Caroba, Boa Esperança do Iguaçu, Bom Jesus do Sul, Capanema, Cruzeiro do Iguaçu, Dois Vizinhos, Éneas Marques, Flor da Serra Do Sul, Francisco Beltrão, Manfrinópolis, Marmeleiro, Nova Esperança do Sudoeste, Nova Prata do Iguaçu, Pérola D'oeste, Pinhal de São Bento, Planalto; Pranchita, Realeza, Renascença, Salgado Filho, Salto do Lontra, Santa Izabel do Oeste, Santo Antônio do Sudoeste, São Jorge D'Oeste, and Verê. We chose this region to develop the study because Paraná is the state that sells the fourth highest amount of pesticides in Brazil.²⁶ This reflects an extensive use of pesticides in the state’s agricultural activities, which play a significant role in the Gross Domestic Product (GDP) of the 27 municipalities that make up this health area.²⁶ More than 50% of the region’s inhabitants engage in agricultural activities, with a particular focus on family farming. This population is subject to considerable pesticide exposure, especially glyphosate, atrazine, and 2,4-dichlorophenoxyacetic (2,4-D), which are widely used in soybean, corn, and wheat monocultures in the region.²⁶ Figure 1A depicts the correlation between breast cancer cases and the amount of pesticides used by municipalities in the Eighth Health Region of Paraná.

Data were grouped into three categories: patient characteristics, tumor characteristics, and disease characteristics (Figure 1B).

2.1.1. Patient characteristics

The following data were used as defining parameters of patient characteristics: age in years at diagnosis and menopausal status at diagnosis, dichotomized as presence and absence. The patient's weight at diagnosis was also obtained in kilograms (kg), height in meters (m), and body mass index (BMI) in kg/m2. The profile of occupational pesticide exposure was obtained using a standardized data collection instrument validated for this purpose.²⁷ The exposure criteria are based on continuous, unprotected, and direct handling of pesticides. Thus, included in the group of pesticide-exposed patients were rural women with a history of direct handling of pesticides without use of protective gloves during preparation and/or dilution of the poisonous solution, application of pesticides, and/or decontamination of personal protective equipment and/or washing of clothes used during spraying, who reported living at least 50% of their lives under direct handling of pesticides at least twice a week for every week of the year. The group of women unexposed to pesticides comprises urban workers with no previous or current history of occupational pesticide exposure.

2.1.2. Tumor characteristics

The following parameters were considered to contain the tumor characteristics: estrogen (ER) and progesterone (PR) receptors' expression profile in percentage (%), considering values greater than zero as positive, and zero as negative; amplification of the epidermal human growth factor receptor 2 (HER2) considering values of "3+" and "2+" with the positive FISH amplification test as positive and values zero, "1+" and "2+" without the FISH amplification test as negative; proliferation index Ki67 in %, considering values below/equal to 14% or above as cut-off (≤14% as low and <14% as high proliferation); molecular subtyping of breast tumors considering the classes Luminal A = any positivity for ER and/or PR and ki67 below/equal to 14%, Luminal B = any positivity for ER and/or PR and ki67 above 14%, HER2-amplified = any ER/PR/ki67 value and presence of amplification for HER2, and Triple Negative = ER/PR/HER2 negative and any ki67 value (as described by the St. Gallen Consensus)²⁸; tumor size represented in mm, histological grade categorized as low (grades 1 and 2) and high (grade 3). Lymph node invasion, presence of angiolymphatic emboli, and occurrence of distant metastases were dichotomized as presence or absence.

2.1.3. Disease characteristics

The parameters considered disease characteristics were stratification of death risk and recurrence (stratified into low risk, intermediate risk, and high risk, as described in Joint Ordinance No. 5 of April 18, 2019),²⁹ chemoresistance development, disease recurrence, and death. All were dichotomized as presence or absence.

2.2. Data analysis

All data wore processed in Python version 3.10.12. A descriptive statistical data analysis was performed, including disease, tumor, and patient characteristics. A ratio was calculated (dividing the frequencies of each molecular subtype) and the differences analyzed by Chi-square and Fisher's exact test for samples with n less than 5. A p>0.05 was considered significant. The parameters that showed significance were subjected to the contingency chi-square test to confirm their independence.

3.1. Breast cancer patients occupationally exposed to pesticides have a prevalence of the Luminal B molecular subtype

Table 1 shows clinicopathological data of the study population according to their pesticide exposure profile. Breast cancer patients (n=386) had a mean age of 56 years, ranging from 22 to 96 years. Average BMI was 27.95 kg/m2 (16.4 – 51.26 kg/m2). About 60% of the patients were occupationally exposed to pesticides. Of this population, 8.25% were stratified into low death risk and recurrence, 55.87% into intermediate risk, and 35.87% were classified as high risk. Regarding the molecular subtype, 33.24% of the patients were classified as Luminal A, 33.8% as Luminal B, 16.62% as HER2-amplified, and 16.34% as Triple-Negative. Also, 28.21% of the tumors were grade 1, 51.68% were grade 2, and 20.11% were grade 3. About 7% of the patients died, 9.36% of the patients had disease recurrence, and 18.97% of the patients developed chemoresistance.

This population was segregated according to the profile of occupational pesticide exposure and presented summary measures similar to those obtained by the total study population (n=386). Breast cancer patients occupationally exposed to pesticides (n=208) had a prevalence of the Luminal B molecular subtype (32.83%), and unexposed patients (n=141) had a prevalence of the Luminal A molecular subtype (37.78%). From the division of the percentages of patients not exposed to pesticides and exposed to pesticides of the Luminal A molecular subtype, it was identified that unexposed patients were 1.2 times more likely to present a molecular subtype associated with a better prognosis of the disease compared to occupationally exposed women. And when dividing the percentages of patients exposed to pesticides and those not exposed to pesticides, the Triple-negative molecular subtype was identified as being 1.5 times more likely for exposed patients to present a molecular subtype associated with a worse prognosis of the disease compared to unexposed women. Exposed patients also have a higher incidence of recurrence (10.19%), chemoresistance (21.26%), and death (7.21%) compared to patients unexposed to pesticides and the total population analyzed, suggesting a worse prognosis.

3.2. Patients exposed to pesticides are more likely to have distant metastasis and affected lymph nodes

We sought to determine which characteristics were significantly distinct in breast cancer patients according to their occupational pesticide exposure profile. Comparisons were exhaustively performed, and only the statistically significant and clinically relevant results were reported here, as summarized in Tables 2 and 3. All variables were evaluated using Chi-square analysis for risk stratification of death and/or recurrence, and molecular subtypes.

Among patients with positive menopause at diagnosis, the number of patients in each molecular subtype was comparatively significant, both for exposed and unexposed patients. Unexposed patients at menopause showed an increase in the frequency of Luminal B patients compared to Triple-negative (p= 0.03 and ratio=2.08) and HER2-amplified (p=<0.01 and ratio=2.78) (Table 3). Exposed patients at menopause showed an increase in the frequency of Luminal B patients compared to Triple-negative (p=0.01 and ratio=1.84) and HER2-amplified (p=<0.01 and ratio=3.29) (Table 2).

Among the tumor characteristics that were significant in the comparison of pesticide exposure are the parameters of distant metastasis and positive lymph node invasion. Breast cancer patients exposed to pesticides were 1.4 times more likely to develop metastasis compared to breast cancer patients unexposed to pesticides. (Figure 2). For the analyses performed on patients exposed to pesticides, an increase in the frequency of Luminal B patients compared to HER2-amplified (p=<0.01 and ratio= 2.31) and Triple-negative (p=<0.01 and ratio=2.73) was observed (Table 2) in the positive subgroup for the distant metastasis parameter. For unexposed patients positive for distant metastasis, there was an increase in the frequency of Luminal B patients compared to Triple-negative (p=0.02 and ratio=3.20) (Table 3), but to a lesser extent. Regarding the lymph node invasion, breast cancer patients exposed to pesticides were 1.3 times more likely of disease spreading compared to breast cancer patients unexposed to pesticides (Figure 2). For the analysis performed on exposed patients belonging to the positive lymph node invasion subgroup, an increase in the frequency of Luminal B patients compared to HER2-amplified molecular subtypes (p=<0.01 and ratio=3.33) (Table 2) was identified. For unexposed patients from the lymph node invasion subgroup, an increase in the frequency of Luminal B patients compared to Triple-negative (p=0.03 and ratio=3.0) was identified (Table 3).

Among the disease characteristics found to be significant, the presence of angiolymphatic emboli were observed. The subgroup of unexposed patients and those positive for the presence of angiolymphatic emboli did not show significant differences in relation to the molecular subtype (Table 3). In the subgroup of exposed patients positive for the presence of angiolymphatic emboli, there was an increase in the frequency of Luminal B patients compared to HER2-amplified (p=0.01 and ratio=3.0) (Table 2).

The parameters menopausal status, distant metastases, lymph node invasion and angiolymphatic emboli were subjected to the contingency chi-square test and confirmed their independence.

Our study indicates that occupational pesticide exposure is linked to the occurrence of breast tumors with more aggressive clinicopathological characteristics. In the exposed population, we observed an increased frequency of disease recurrence, chemoresistance to treatment, death, and predominance of the molecular subtype Luminal B. To the best of our knowledge, this is the first study that uses the Chi-square test for independence and Fisher's exact test for samples with n less than 5 to predict the relationship of variables related to breast cancer severity in a population categorized according to their pesticide exposure profile.

We identified that unexposed women were 1.2 times more likely to have a molecular subtype associated with a better prognosis of the disease compared to women occupationally exposed to pesticides. The most prevalent molecular subtype in these patients was Luminal A, which has slow-growing characteristics characterized by low rates of ki67 proliferation.³⁰ On the other hand, in pesticide-exposed women, we observed they were 1.5 times more likely to have the triple-negative molecular subtype, characterized by more aggressive clinical behavior.³⁰ This trend towards a worse prognosis of women exposed to pesticides was also observed in a toxicoproteomics study conducted by Pizzatti et al. (2020)³¹ and another by Scandolara et al. (2022),³ corroborating that pesticide-exposed women may have a poor prognosis for breast cancer.

In the exploratory analysis, a higher prevalence of patients with the Luminal B molecular subtype (33.8%) was identified, followed by a minimal difference between Luminal A patients (33.24%). This small difference in frequency between the Luminal subtypes found in the study population differs from the percentage found in the populations of China (65.3% Luminal A and 19% Luminal B)³² and the United States (55% Luminal A and 17% Luminal B),³³ countries that are among world leaders in the use of pesticides along with Brazil.³⁴ Although the population of these countries faces similar pesticide exposure as the Brazilian population, one probable reason why a prevalence of more aggressive molecular subtypes is not observed is the lack of studies that segregate the exposed population for a more detailed evaluation. This inclination towards the emergence of tumors with molecular subtypes with an unfavorable prognosis may be related to the promotion of cell proliferation induced by pesticide exposure.^35,36

Pesticide exposure is known to be associated with dysregulation of immune and inflammatory responses.³⁷ The study conducted by Silva et al. (2022)⁹identified a predominance of intermediate death risk and recurrence for women who were exposed to pesticides. Due to its uncertain characteristics, intermediate risk may imply the recurrence of disease or even systemic damage and has been linked to poor immunological profiles. Other studies have reported immune deregulation in women exposed to pesticides,^38,39 which may favor the development of more aggressive tumors. For example, failure to produce Th1 immune responses can influence the development of large tumor masses in the long term, potentially resulting in aggressive tumor behaviors, such as the occurrence of metastases observed in the exposed patients described in this study.⁴⁰

Luminal B breast cancer patients exposed to pesticides were more likely to develop lymph node invasion and distant metastasis than other subtypes. Comparatively, we observed an increased ratio when compared to samples from other molecular subtypes. Li et al. (2019)⁴¹ suggested that lymph node involvement and tumor metastasis have a strong association with pathogenic alterations in TP53 expression. These modifications were observed with a higher mutagenic frequency in patients exposed to pesticides in the study conducted by Scandolara et al. (2022),³ demonstrating the cascade of progressive damage generated by pesticide exposure, including oncogenesis.

Further aggravating harm to the health of the population exposed to pesticides, a study conducted on a population chronically exposed to pesticides—even at low exposure doses—found an accumulation of DNA lesions due to failures in the genetic material repair system.⁴² Thus, the accumulation of mutations observed in the immune and inflammatory responses triggered by pesticide exposure,^3,31,38,39which generally result in the inactivation of tumor suppressor genes, are indications of genomic instability.⁴³ This genomic instability increases susceptibility to metastasis development.⁴⁴ Song et al. (2011)⁴⁵ found a greater predisposition to lymph node involvement in patients with angiolymphatic emboli, indicating a greater tendency to a worse prognosis. However, studies on the influence of the presence of angiolymphatic emboli on breast cancer are still scarce. In addition, due to the hypoxic environment that the presence of angiolymphatic emboli provides, some authors consider it as a precursor in the development of metastasis in cancer patients,^37,46,47reinforcing the idea of a greater tendency to an unfavorable prognosis for the patient, especially in the context of pesticide exposure.

Pesticides are known endocrine disruptors and can influence the development of tumors in the female reproductive system,²⁵ increase aromatase activity and estrogen production,^48,49 reduce fertility,⁴⁸ augment estrogen production,⁵⁰ increase androgen availability,^51,52 competitively bind to estrogen cell receptors,⁵³ enhance proliferation of estrogen-sensitive cells, and inhibit corticosterone synthesis in the adrenal cortex. ^48,51,54 In this context, we identify 1.8 times more likely of exposed patients having menopause at diagnosis compared to unexposed patients.

Pizzatti et al. (2020)³¹ reported that pesticide-exposed patients in menopause had significantly reduced levels of tumor necrosis factor-alpha (TNF-α) when compared to unexposed patients, suggesting that exposure may affect the production of TNF-α in the absence of estrogen, resulting in worsening of the disease due to the failure of antitumor mechanisms. This could help to explain the higher incidence of the triple-negative molecular subtype in the population exposed to pesticides.

This study has limitations, including the modest sample size and the lack of other risk factors, such as dietary habits and lifestyle. Its strength is that the correlational analysis of pesticide exposure with clinicopathological parameters of breast cancer may be influencing the worse prognosis found in patients living in southwestern Paraná. Therefore, we reiterate the urgency of discussing and changing policies regulating the use of pesticides and the need for screening exposed populations at risk of developing more aggressive disease.

Data availability statement

The original contributions presented in the study are included in the article Materials and Methods. Further inquiries can be directed to the corresponding author. Data, including unique materials, documentation, and codes used in the analysis, are available at https://github.com/Laboratorio-de-Analise-de-Dados/Article_pesticide_exposure.

Ethics statement

The studies involving humans were approved by the Research Ethics Committee (CEP) of the State University of Western Paraná (UNIOESTE) under the number

CAAE 35524814.4.0000.0107. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

Author Contributions:

Conceptualization: ICC, CP, and GFS

Methodology: ICC, RFA, LLS, MPAB, SCG, LZPC, DR, CP, and GFS

Investigation: ICC, RFA, LLS, MPAB, SCG, LZPC, DR, CP, and GFS

Visualization: ICC, CP, and GFS

Supervision: CP and GFS

Writing—original draft: ICC

Writing—review & editing: ICC, DR, CP, and GFS

Funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. Part of this research was supported by Carlos Chagas Institute, Fiocruz/PR, and by Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq.

Conflict of interest:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations or those of the publisher, the editors, and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

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Table 1 - Clinicopathological characteristics of the patients included in the study.

	Total population	All patients (n)	Exposed, frequency	Exposed, number (n)	Unexposed, frequency	Unexposed, number (n)
Patient Characteristics
Mean age at diagnosis (years)	56	353	56	197	56	134
BMI (kg/m²)	27.95	259	27.82	150	27.92	97
Occupational pesticide exposure	59.6%	208	100.0%	208	0.0%	0
Tumor Characteristics
Average tumor size (mm)	29,21	357	30,82	201	28,11	134
Molecular subtyping
Luminal A	33.24%	118	30.81%	61	37.78%	51
Luminal B	34.37%	122	33.33%	66	34.81%	47
HER2-amplified	16.62%	59	16.67%	33	15.56%	21
Triple-negative	15.77%	56	19.19%	38	11.85%	16
Grade 1	28.21%	101	29.15%	58	29.2%	40
Grade 2	51.68%	185	52.26%	104	48.91%	67
Grade 3	20.11%	72	18.59%	37	21.9%	30
Disease characteristics
Stratification of death risk and recurrence
Low	8.25%	26	8.24%	15	8.26%	10
Intermediate	55.87%	176	52.2%	95	60.33%	73
High	35.87%	113	39.56%	72	31.4%	38
Recurrence	9.36%	35	10.19%	21	9.93%	14
Chemoresistance	18.97%	70	21.26%	44	17.27%	24
Death	7.01%	27	7.21%	15	7.09%	10

The parameters age at diagnosis (years), BMI (kg/m²), and tumor size are presented by the population mean analyzed, and the parameters occupational pesticide exposure, molecular subtyping, histological grade, stratification of death risk and recurrence, recurrence, and chemoresistance are presented as a percentage of the analyzed group in relation to the total number of individuals in the population. The number of patients in each variable is different due to missing values (all variables have less than 15% of missing values, except for the variable BMI with 28%). n represents the number of patients evaluated for each parameter.

Table 2 - Frequency, ratio and p-value of the patients exposed to pesticides for molecular subtypes.

		Molecular subtype
	Total	Lum A	Lum B	HER2-amplified	Triple-negative	Ratio Lum B/ Lum A	p-value	Ratio Lum B / HER2-amplified	p-value	Ratio Lum B/Triple-negative	p-value
Estrogen receptor expression	133	58	65	10	0	1.12	0.53	6.50	<0.01	0	<0.01
Progesterone receptor expression	89	43	38	8	0	0.88	0.58	4.75	<0.01	0	<0.01
HER2-amplified	24	0	0	24	0	0.00	NA	0.00	<0.01	0	NA
KI67 (%)	120	8	66	27	19	8.25	<0.01	2.44	<0.01	3.47	<0.01
Presence of angiolymphatic emboli	52	15	18	6	13	1.20	0.6	3.00	0.01	1.38	0.37
Lymph node invasion	68	19	30	9	10	1.58	0.12	3.33	<0.01	3.00	<0.01
Distant metastasis	76	22	30	13	11	1.36	0.27	2.31	<0.01	2.73	<0.01
Menopausal status	127	42	46	14	25	1.10	0.67	3.29	<0.01	1.84	0.01
Pesticide exposure	198	61	66	33	38	1.08	0.66	2.00	<0.01	1.74	<0.01
Chemoresistance	44	8	15	10	11	1.88	0.14	1.50	0.32	1.36	0.43
Recurrence	21	4	7	5	5	1.75	0.54^α	1.40	0.56	1.40	0.56
Death	15	2	3	2	8	1.50	1.00^α	1.50	1.00^α	0.38	0.02^α

A ratio was calculated (dividing the frequencies of each molecular subtype) and the differences analyzed by Chi-square and Fisher's exact test for samples with n less than 5. A p<0.05 was considered significant. The acronym Lum corresponds to the molecular subtype Luminal. NA represents when the calculation was not applicable. ^α represents that p-values were calculated using Fisher's exact test, the other values were calculated using the chi-square test for independence.

Table 3 - Frequency, ratio and p-value of the patients unexposed to pesticides for molecular subtypes.

			Molecular subtype
	Total (n)		Lum A		Lum B		HER2-amplified		Triple-negative		Ratio Lum B/ Lum A		P-value		Ratio Lum B / HER2-amplified		P-value		Ratio Lum B /Triple-negative		P-value
Estrogen receptor expression		106		51		47		8		0		0.92		0.69		5.88		<0.01		0		<0.01
Progesterone receptor expression		78		35		36		7		0		1.03		0.91		5.14		<0.01		0		<0.01
HER2-amplified		18		0		0		18		0		0.00		NA		0.00		<0.01		0		NA
KI67 (%)		84		6		46		18		14		7.67		<0.01		2.56		<0.01		3.29		<0.01
Presence of angiolymphatic emboli		31		7		16		4		4		2.29		0.06		4.00		0.26 ^α		4.00		0.75^α
Lymph node invasion		32		9		15		3		5		1.67		0.22		5.00		0.73^α		3.00		0.03
Distant metastasis		35		10		16		4		5		1.60		0.24		4.00		0.53^α		3.20		0.02
Menopausal status		85		39		25		9		12		0.64		0.08		2.78		<0.01		2.08		0.03
Pesticide exposure		0		0		0		0		0		0.00		NA		0.00		NA		0.00		NA
Chemoresistance		23		6		11		3		3		1.83		0.23		3.67		0.52^α		3.67		1.00^α
Recurrence		14		4		5		2		3		1.25		0.73^α		2.50		1.00^α		1.67		0.41^α
Death		8		0		2		0		6		0.00		0.23^α		0.00		1.00^α		0.33		0.00^α

No competing interests reported.

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Occupational pesticide exposure is linked to the prevalence of Luminal B breast cancer and poor prognosis features in Brazilian rural women

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