Influence of CYP19A1 gene expression levels in women with breast cancer: a systematic review of the literature.

Breast cancer is the most frequently diagnosed malignant neoplasm in women and is considered a multifactorial disease of unknown etiology. One of the major risk factors is genetic alteration. Changes in CYP19A1 gene expression levels have been associated with increased risk and increased aggressiveness of breast cancer. Increased CYP19A1 gene expression and/or aromatase activity are among the major regulatory events for intratumoral production of estrogens in breast malignant tissues. This systematic review aimed to investigate the influence of CYP19A1 gene expression levels in women with breast cancer. The research was carried out using the PubMed, Scopus, and Web of Science databases. Searches were conducted between February 2 and May 15, 2019. Inclusion criteria were studies published between 2009 and 2019, English language publications, and human studies addressing the gene expression of CYP19A1 in breast cancer. A total of 6.068 studies were identified through PubMed (n=773), Scopus (n=2,927), and the Web of Science (n=2,368). After selecting and applying the inclusion and exclusion criteria, six articles were included in this systematic review. This systematic review provides evidence that increased or decreased levels of CYP19A1 gene expression may be related to pathological clinical factors of disease, MFS, OS, DFS, WATi, markers of metabolic function, concentrations of E1, FSH, and in the use of multiple exons 1 of the CYP19A1 gene in breast cancer.

Evaluation of vitamin D receptor expression in uterine leiomyoma and nonneoplastic myometrial tissue: a cross-sectional controlled study.

BACKGROUND: Uterine myomas are the most frequent benign solid pelvic tumors in women of reproductive age. At present, uterine myomas are the most common indication for hysterectomy because of the morbidity they cause, including intense bleeding, compression of adjacent organs, pain, and infertility. Some studies show that vitamin D receptor (VDR) expression is correlated with the etiology of uterine leiomyomas. This study aimed to assess the expression of VDR in uterine leiomyoma and nonneoplastic myometrial tissue. METHODS: A controlled cross-sectional study involving 40 women who underwent abdominal hysterectomy the Department of Gynecology of the Getúlio Vargas Hospital of Federal University of Piauí, Brazil, was performed to compare the immunohistochemical expression of VDR in samples of uterine leiomyoma tissue with adjacent nonneoplastic myometrial tissue. The mean percetages of stained nuclei in the two groups was compared by Student's t teste, with significance established at p < 0.05. RESULTS: The percentage of cells with nuclei stained by anti-VDR in the myometrial and leiomyoma tissue was 79.52 % (± 4.32) and 60.22 % (± 7.24), respectively (p < 0.0001). CONCLUSIONS: The mean percentage of nuclei expressing VDR was significantly lower in the uterine leiomyoma than in nonneoplastic myometrial tissue.

Influence of CYP19A1 gene expression levels in women with breast cancer: a systematic review of the literature

Breast cancer is the most frequently diagnosed malignant neoplasm in women and is considered a multifactorial disease of unknown etiology. One of the major risk factors is genetic alteration. Changes in CYP19A1 gene expression levels have been associated with increased risk and increased aggressiveness of breast cancer. Increased CYP19A1 gene expression and/or aromatase activity are among the major regulatory events for intratumoral production of estrogens in breast malignant tissues. This systematic review aimed to investigate the influence of CYP19A1 gene expression levels in women with breast cancer. The research was carried out using the PubMed, Scopus, and Web of Science databases. Searches were conducted between February 2 and May 15, 2019. Inclusion criteria were studies published between 2009 and 2019, English language publications, and human studies addressing the gene expression of CYP19A1 in breast cancer. A total of 6.068 studies were identified through PubMed (n=773), Scopus (n=2,927), and the Web of Science (n=2,368). After selecting and applying the inclusion and exclusion criteria, six articles were included in this systematic review. This systematic review provides evidence that increased or decreased levels of CYP19A1 gene expression may be related to pathological clinical factors of disease, MFS, OS, DFS, WATi, markers of metabolic function, concentrations of E1, FSH, and in the use of multiple exons 1 of the CYP19A1 gene in breast cancer.

Human Epidermal Growth Factor Receptor-2 gene polymorphism and breast cancer risk in women from the Northeastern region of Brazil.

OBJECTIVES: In the Human Epidermal Growth Factor Receptor-2 (HER2) rs1136201 variant, the presence of the G allele may promote cellular alterations and increase breast cancer risk, in addition to enhanced cellular proliferation, tumor aggressiveness, and metastases. The aim of this study was to investigate the presence of the single-nucleotide polymorphism (SNP) variant, rs1136201, within the HER2 gene in women from the Northeastern region of Brazil and breast cancer risk. METHODS: The study included 140 women who were divided into two groups, case (breast cancer) and control (without breast cancer), with 70 women in each group. Peripheral blood of each woman was drawn for the study of genomic Deoxyribonucleic acid (DNA) extracted from leukocytes using the genotyping technique by real-time polymerase chain reaction. RESULTS: The GG genotype occurred in 1 woman in both groups (1.4%) (p=0.32), while the AG genotype occurred in 19 (27.2%) and 13 (18.6%) women in the case and control (p=1.00) groups, respectively. No statistically significant difference in GG and AG genotypes was observed between the case and control groups in premenopausal women (p=1.00). Furthermore, no significant difference in genotypes was observed between the groups, among postmenopausal women (p=0.14). CONCLUSION: In this study, the HER2 rs1136201 polymorphism did not show any statistically significant association with breast cancer, both in premenopausal and postmenopausal women. Nevertheless, further studies with a larger sample size should be performed to assess the association of HER2 polymorphism with breast cancer risk in women from the Northeastern region of Brazil.

Assessment of IGF-1 expression in the peripheral blood of women with recurrent breast cancer.

Breast cancer is the most common malignancy affecting women worldwide. The insulin-like growth factor 1 (IGF-1) gene encodes a protein responsible for a wide variety of physiological processes, including differentiation and cell proliferation. Despite several studies on tumor tissues, no study has evaluated IGF-1 expression in the peripheral blood of women with recurrent breast cancer.In this cross-sectional study, IGF-1 expression in the peripheral blood of 146 women with breast cancer treated approximately 5 years ago was quantified by quantitative reverse transcription polymerase chain. The women were divided into 2 groups: non-recurrence (n = 85) and recurrence (n = 61). Statistical analysis of the data was performed using ANOVA, Mann-Whitney, and Chi-squared tests (P < .05).The results showed no significant difference in IGF-1 expression between the non-recurrence and recurrence groups (P = .988). In the subgroups of patients with lymph node involvement, no statistically significant difference was observed in IGF-1 expression between women with recurrence and those non-recurrence (P = .113). In patients without lymph node metastases, IGF-1 messenger ribonucleic acid (mRNA) expression levels were significantly higher in the non-recurrence group than in the recurrence group (P = .019). Furthermore, using the median IGF-1 mRNA expression as the cutoff point, it was obtained a statistically significant difference in tumor histological grade among women with recurrent breast cancer (P = .042).These data showed significantly higher IGF-1 expression in women without lymph node metastases in the non-recurrence group compared with the recurrence group. In addition, a significant difference was observed in median IGF-1 mRNA expression in relation to tumor histological grade in women with recurrent breast cancer.

Expression of Nrf2 and NF-κB transcription factors in breast cancer and breast fibroadenoma: Insights for a new therapeutic approach.

Background: Cancer and fibroadenoma are the most common breast tumors in women of reproductive age. Nuclear factor erythroid 2-related factor 2 (Nrf2) and the nuclear factor kappaB (NF-κB) transcription factor play an important role in the inflammatory process and in cell proliferation. However, few studies have analyzed these markers in breast cancer and fibroadenoma in women of reproductive age. Results: Light microscopy showed a higher concentration of anti-Nrf2 and anti-NF-κB-stained nuclei in breast cancer than in fibroadenoma. The mean percentage of stained nuclei for Nrf2 was 7.12 ± 5.2 and 43.21 ± 19.83 in the control and study groups, respectively (p < 0.0001). The mean percentage of anti-NF-κB was 10.75 ± 7.09 and 56.14 ± 21.19 (mean ± standard deviation) in the control and study groups, respectively (p < 0.0001). Histological grade 3 tumors showed a significantly higher expression of Nrf2 and NF-κB than grade 1 tumors (p < 0.05). Material and methods: This study was approved by the Institutional Review Board of Federal University of Piaui and all patients assigned an inform consent term prior to the study initiation. Nrf2 and NF-κB expression was evaluated by immunohistochemistry in 66 patients, divided into two groups, control (fibroadenoma, n = 36) and study (cancer, n = 30). The data were analyzed using ANOVA test and the statistical significance was established at p < 0.05. Conclusion: Nrf2 and NF-κB expression was significantly higher in breast cancer than in fibroadenoma, in addition to having a greater association with more aggressive tumors.

CYP19A1 gene expression in the peripheral blood of Brazilian women with breast cancer relapse.

BACKGROUND: The CYP19A1 gene, which encodes the enzyme responsible for androgen aromatization into estrogens, may play an important role in breast cancer aggressiveness. However, no study has evaluated CYP19A1 gene expression in the peripheral blood of women with relapsed breast cancer. METHODS: In this cross-sectional study, CYP19A1 gene expression was quantified by RT-PCR in the peripheral blood of 146 women with breast cancer who were first divided into two groups according to the expression of CYP19A1 (low and high); each group had 73 patients. Subsequently, women were divided into two groups: those without recurrence (control, n = 85) and those with recurrence (study, n = 61). Statistical analysis of the data was performed using ANOVA, the Mann-Whitney, Chi-square or Fisher's exact test (p <  0.05). RESULTS: There were no significant differences between the relative expression of CYP19A1 mRNA in the low expression group and the high expression group according to the variables studied. There were no significant differences in CYP19A1 gene expression in the study and control groups (p = 0.8461). In the relapse group, CYP19A1 gene expression was significantly higher in the hybrid luminal subtype than in the triple-negative subtype (p = 0.0321), whereas it was significantly lower in HER2-negative cases than in HER2-positive cases (p <  0.0376). Women with locoregional recurrence showed higher expression than women with distant recurrence (p <  0.0001). CONCLUSIONS: The present study found no significant differences between women with high and low expression of the CYP19A1 gene mRNA or between those in the study group and the control group. However, in women with recurrence, there was increased expression of CYP19A1 mRNA in those who had the luminal hybrid subtype and locoregional relapse and decreased expression in those negative for HER2.

Human Epidermal Growth Factor Receptor-2 gene polymorphism and breast cancer risk in women from the Northeastern region of Brazil

OBJECTIVES: In the Human Epidermal Growth Factor Receptor-2 (HER2) rs1136201 variant, the presence of the G allele may promote cellular alterations and increase breast cancer risk, in addition to enhanced cellular proliferation, tumor aggressiveness, and metastases. The aim of this study was to investigate the presence of the single-nucleotide polymorphism (SNP) variant, rs1136201, within the HER2 gene in women from the Northeastern region of Brazil and breast cancer risk. METHODS: The study included 140 women who were divided into two groups, case (breast cancer) and control (without breast cancer), with 70 women in each group. Peripheral blood of each woman was drawn for the study of genomic Deoxyribonucleic acid (DNA) extracted from leukocytes using the genotyping technique by real-time polymerase chain reaction. RESULTS: The GG genotype occurred in 1 woman in both groups (1.4%) (p=0.32), while the AG genotype occurred in 19 (27.2%) and 13 (18.6%) women in the case and control (p=1.00) groups, respectively. No statistically significant difference in GG and AG genotypes was observed between the case and control groups in premenopausal women (p=1.00). Furthermore, no significant difference in genotypes was observed between the groups, among postmenopausal women (p=0.14). CONCLUSION: In this study, the HER2 rs1136201 polymorphism did not show any statistically significant association with breast cancer, both in premenopausal and postmenopausal women. Nevertheless, further studies with a larger sample size should be performed to assess the association of HER2 polymorphism with breast cancer risk in women from the Northeastern region of Brazil.

The effect of raloxifene on Bax protein expression in breast carcinomas of postmenopausal women.

The actual role of Bax protein as a prognostic biomarker that predicts response to therapy remains unclear. The objective of this study was to evaluate the effect of raloxifene on Bax protein expression in breast carcinomas of postmenopausal women. Twenty postmenopausal patients with operable stage II, estrogen receptor-positive, infiltrating ductal breast carcinoma were treated with oral raloxifene at a dose of 60 mg/day for a period of 28 days before definitive surgery. Tumor samples were obtained by incisional biopsy at the time of diagnosis and again at the time of definitive surgical treatment. Immunohistochemical evaluation of Bax expression was assessed semiquantitatively based on the fraction of stained tumor cells and intensity of staining. McNemar's test was used to analyze data (P < 0.05). Eleven of the 20 (55%) patients were classified as positive for Bax expression before raloxifene treatment, whereas 9(45%) were classified as positive after raloxifene treatment (P = 0.479). In conclusion, raloxifene did not alter Bax expression significantly in estrogen receptor-positive breast carcinomas of postmenopausal women.

Assessment of micronucleus frequency in the peripheral blood of female rats in persistent estrus treated with selective estrogen receptor modulators.

The aim of this study was to evaluate micronucleus (MN) frequency in polychromatic erythrocytes (PCE) of female rats in persistent estrus (a model developed to mimic polycystic ovary syndrome) treated with selective estrogen receptor modulators (SERMs, tamoxifen, and raloxifene). Forty female Wistar-Hannover rats were divided into four groups of 10 animals each: Group I (normally cycling rats) and Group II (persistent estrus) both received only vehicle, while Group III (persistent estrus) was treated with tamoxifen (250 µg/animal/day) and Group IV (persistent estrus) was treated with raloxifene (750 µg/animal/day). Tamoxifen and raloxifene were given by oral gavage beginning on postnatal day 90 and continuing for 30 consecutive days. Peripheral blood samples were collected from tails 1 day following the last exposure. Blood smears were made on glass slides and stained with 10% Giemsa solution. ANOVA and a Tukey post-hoc test were used for data analysis. Mean percentages of MN were 1.82 ± 0.13, 5.20 ± 0.24, 3.32 ± 0.13, and 3.04 ± 0.12 in Groups I, II, III, and IV, respectively. The results indicate that tamoxifen and raloxifene similarly reduced the formation of MNPCE of female rats in persistent estrus (P < 0.0001 for Groups III and IV vs. Group II), using the dosages and time periods applied in the present study. The data suggest possibly antimutagenic effects of SERMs under high levels of estrogens. The findings also suggest that this is an interesting animal model for studying the genotoxicity of estrogens.

Comparative study of tamoxifen and raloxifene on endometrial cell proliferation of female rats in persistent estrus.

OBJECTIVE: The objective of the study was to compare the effect of tamoxifen and raloxifene on the endometrium of female rats in persistent estrus, by Ki-67 protein expression. METHODS: The study comprised 60 Wistar-Hannover female rats in persistent estrus, induced by a single subcutaneous dose of 1.25 mg of testosterone propionate on the second day of age. At 90 days of life, the animals were randomly divided into 3 groups of 20 animals each. Group 1 (control), received only placebo; group 2, the animals were treated with tamoxifen, 250 µg/d; and group 3, the rats were treated with 750 µg/d of raloxifene by gavage during 30 days. Then, the animals were killed, and the endometrium was removed for immunohistochemical analysis of Ki-67 antigen expression. Statistical analysis was performed by ß regression model (P < 0.05). RESULTS: Mean percentages of Ki-67 protein expression in the endometrium of rats in persistent estrus were 43.21% ± 3.39%, 7.36% ± 0.95%, and 7.20% ± 0.76% in groups 1, 2 and 3, respectively (P < 0.001). There was no statistical difference between groups 2 and 3 (P = 0.7159). CONCLUSIONS: The present results indicate that, at the doses and during the time of treatment used, both tamoxifen and raloxifene induce atrophy in a similar way of endometrial epithelium of rats in persistent estrus.

Evaluation of arterial blood pressure and renal sodium handling in a model of female rats in persistent estrus.

The aim of this study was to evaluate the arterial blood pressure and renal function of female rats in persistent estrus. Twenty-five female Wistar-Hannover rats were randomly divided into two groups: Group I (control, n = 15) and experimental (persistent estrus, n = 10). A tail-cuff system was used to measure blood pressure at 12 weeks of life. Parameters to evaluate renal function were taken into consideration. A significant increase in arterial pressure and a significant decrease in fractional sodium excretion were found in the androgenized animals compared to controls. There was no difference between the two groups with respect to glomerular filtration rate or in fractional potassium excretion. An increase in blood pressure and a reduction in fractional sodium excretion occur in female rats in persistent estrus.

Immunohistochemical expression of E-cadherin in sclerosing adenosis, ductal carcinoma in situ and invasive ductal carcinoma of the breast.

E-cadherin (EC) is an important glycoprotein cell-adhesion molecule that appears to play a significant role in the progression of breast lesions. The objective of this study was to evaluate EC expression in sclerosing adenosis, ductal carcinoma in situ and invasive ductal carcinoma. Samples of breast lesions from 44 women were used in this study, comprising cases of sclerosing adenosis (n = 11), ductal carcinoma in situ (DCIS) (n = 10) and invasive ductal carcinoma (n = 23). Immunohistochemical evaluation of EC expression was assessed semiquantitatively and considered negative (<10% of cells with stained cytoplasmic membranes), positive+ (10-50% of cells stained) or positive++ (> 50% of cells stained). Fisher's exact test was used to compare the distribution of staining intensity in the lesions (P< 0.05). There was a progressive loss of EC expression from benign to malignant lesions. This difference was statistically significant when sclerosing adenosis was compared with DCIS (P < 0.0002), when sclerosing adenosis was compared with invasive ductal carcinoma (P < 0.008) and when DCIS was compared with invasive ductal carcinoma (P < 0.007). The present findings point to a significant association between reduced EC expression and the progression and aggressivity of breast lesions. Diagn. Cytopathol. 2010. (c) 2009 Wiley-Liss, Inc.

Effects of raloxifene on Ki-67 and CD34 antigen expression in breast cancer.

BACKGROUND: The aim of this study was to evaluate the effect of raloxifene on CD34 and Ki-67 antigen expression in breast cancer specimens from postmenopausal women. METHODS: Sixteen postmenopausal patients with operable, stage II (>or=3 cm), estrogen receptor-positive breast cancer, who took 60 mg of raloxifene daily for 28 days, participated in this study. Immunohistochemistry was carried out in tumor samples prior to and following raloxifene treatment to evaluate CD34 and Ki-67 protein expression. Angiogenesis was quantified in 10 randomly selected fields per slide, and Ki-67-stained nuclei were counted in 1,000 cells per slide using an image capture and analysis system with 400x magnification. Student's t test for paired samples was used for the statistical analysis of data. Statistical significance was established at p < 0.05. RESULTS: The mean number of microvessels was 44.44 +/- 3.54 prior to raloxifene therapy and 22.63 +/- 1.61 following therapy (p < 0.001), and the mean percentage of Ki-67-stained nuclei was 19.28 +/- 1.61 and 12.13 +/- 1.48 prior to and following raloxifene treatment, respectively (p < 0.001). CONCLUSION: Raloxifene significantly reduces CD34 and Ki-67 protein expression in breast carcinoma in postmenopausal women.

Evaluation of Ki-67 antigen expression in the zona reticularis of the adrenal cortex of female rats in persistent estrus.

BACKGROUND: Hyperandrogenism and chronic anovulation are basic characteristics of polycystic ovary syndrome (PCOS), and androgens from the adrenal glands play an important role in the hyperandrogenism. Our aim was to evaluate the proliferative activity in the zona reticularis cells of the adrenal cortex of female rats in persistent estrus, a model developed to mimic PCOS. METHODS: Forty-four female Wistar-Hannover rats were randomly divided into two groups: control (n = 17) and animals which received 1.25 mg testosterone propionate s.c. on the second day of life (n = 27). At 90 days of age, after confirmation of persistent estrus, the animals were sacrificed, and the adrenal glands were removed and fixed in 10% buffered formalin to investigate Ki-67 antigen (marker of proliferation) expression by immunohistochemical analysis. Student's t-test and Levene's test were used in the statistical analysis. RESULTS: The mean percentage of Ki-67-stained nuclei per 1000 cells in the zona reticularis of the adrenal cortex was 15.58 +/- 1.14 (SEM) and 51.59 +/- 1.81 in the control and persistent estrus animals, respectively (P < 0.001). CONCLUSIONS: Proliferative activity in the zona reticularis cells of the adrenal cortex of the androgenized female rats was significantly greater than that of the control animals.

Embryonal rhabdomyosarcoma of the uterus associated with uterine inversion in an adolescent: a case report and published work review.

Embryonal rhabdomyosarcomas of the uterus are extremely rare malignant tumors and their association with uterine inversion is even rarer. The present case report refers to a 15-year-old Brazilian girl admitted to hospital with a 3-month history of transvaginal bleeding and a mass protruding from the vaginal introitus. Following biopsy of the lesion and confirmation of the diagnosis of embryonal rhabdomyosarcoma, the patient was submitted to total abdominal hysterectomy followed by chemotherapy and radiotherapy. The patient died 9 months after surgery. Embryonal rhabdomyosarcoma of the uterus is very rare and extremely aggressive. According to a published work review, there has been only one report to date on the association of embryonal rhabdomyosarcoma of the uterus and uterine inversion.

Cavernous hemangioma in the axillary region mimicking malignant neoplasia.

Cavernous hemangiomas of the breast are rare and when present, are generally well defined. Their presentation in the axilla mimicking cancer is extremely rare. This report describes the case of a premenopausal woman with a cavernous hemangioma in the axillary region with clinical, mammographic, and sonographic characteristics strongly suggestive of malignancy. The definitive diagnosis was based on histological analysis of the lesion complemented by immunohistochemistry for the CD34 marker. Cavernous hemangioma in the axillary region is extremely rare and the possibility of its presentation in this location mimicking cancer should be considered.

Primary twin omental pregnancy: report of a rare case and literature review.

OBJECTIVE: To report a very rare case of a primary twin omental pregnancy. DESIGN: Case report. SETTING: The emergency department of a university teaching hospital. PATIENT(S): A 36-year-old woman. INTERVENTION(S): Partial omentectomy by laparotomy. MAIN OUTCOME MEASURE(S): Laparotomy successfully performed in the case of a twin omental pregnancy. RESULT(S): A 36-year-old woman presented with intense abdominal pain of 3 days' duration, hypotensive, with distended abdomen and signs of peritoneal irritation, and no transvaginal bleeding or evidence of topic pregnancy. Laparotomy showed normal internal genital organs and the presence of a large hemoperitoneum with a twin omental pregnancy. Partial omentectomy was performed. The patient progressed well postoperatively, and subsequent beta-hCG titer was negative. CONCLUSION(S): Primary omental twin pregnancy is extremely rare, and when associated with acute abdomen and large hemoperitoneum, laparotomy is required for treatment.

Morphometric analysis of the urethra of castrated female rats treated with tamoxifen.

OBJECTIVES: The aim of this study was to evaluate the effects of tamoxifen on the weight and thickness of the urethral epithelium of castrated female rats. METHODS: Forty castrated adult female Wistar-Hannover rats were randomly divided into two groups: Group I (n=20) in which the animals received only the vehicle (propylene glycol) and Group II (n=20) in which the rats received tamoxifen 250microg/day by gavage. After 30 days of treatment, all animals were sacrificed and the urethra was immediately removed for weighing. Next, the urethra was divided into the proximal and distal segments, which were fixed in 10% formaldehyde and submitted to routine histological techniques for morphometric study. The data were analyzed using the weighted minimum mean-square error method and Student's t-test for two independent samples (p<0.05). RESULTS: There was a significant increase in the mean weight of the urethra in the rats of Group II compared to the control group, 32.0+/-2.0mg and 22.0+/-1.6mg, respectively (p<0.001). The mean thickness of the distal urethral epithelium of the animals treated with tamoxifen was significantly greater than that of the control group, 42.8+/-2.0microm and 36.6+/-1.5microm, respectively (p<0.001). There was no statistically significant difference between the two groups with respect to the epithelial thickness of the proximal urethra (p=0.514). CONCLUSION: Treating castrated adult rats with 250microg/day of tamoxifen for 30 days may increase the weight of the urethra and the thickness of the distal urethral epithelium.

Multiparity after an initial diagnosis of Parkinson's disease: a report on a rare case.

OBJECTIVE: To report a very rare case of multiparity after a diagnosis of Parkinson's disease (PD). DESIGN: Case report. SETTING: Department of neurology and gynecology of a university teaching hospital. PATIENT(S): A 36-year-old multiparous woman. INTERVENTION(S): Treatment of PD during pregnancy. MAIN OUTCOME MEASURE(S): The effect of multiparity on PD. RESULT(S): Multiparity had no effect on the progression of PD, and treatment of the disease during pregnancy appears to have been safe. CONCLUSION(S): Multiparity after a diagnosis of PD is extremely rare and, in the present case, had no effect on the progression of the disease.