Clinical follow up of Mexican women with early onset of breast cancer and mutations in the BRCA1 and BRCA2 genes

Estudio de seguimiento clínico de mujeres mexicanas con cáncer de mama de inicio temprano y mutaciones en los genes BRCA1 y BRCA2

Ana Laura Calderón-Garcidueñas, MD, MSc^{I, II}; Pablo Ruiz-Flores, MD, PhD^III; Ricardo M Cerda-Flores, PhD^II; Hugo A Barrera-Saldaña, PhD^IV

^IDepartment of Pathology, Hospital de Especialidades No. 25, Instituto Mexicano del Seguro Social (IMSS), Monterrey, Nuevo León, México
^IIDepartment of Population Genetics, Northeastern Center for Biomedical Research, IMSS, Monterrey, Nuevo León, México ]]> ^IIICenter for Biomedical Research, Department of Genetics, Medical School, Autonomous University of Coahuila. Torreón, Coahuila
^IVDepartment of Biochemistry, Medical School, Universidad Autonóma de Nuevo León. Monterrey, Nuevo León, México

ABSTRACT

OBJECTIVE: This study describes the presence of mutations in BRCA1 and BRCA2 genes in a group of Mexican women and the clinical evolution of early onset breast cancer (EOBC).
MATERIAL AND METHODS: A prospective hospital-based study was performed in a sample of 22 women with EOBC (7 in clinical stage IIA, 8 in IIB, and 7 in IIIA). The patients attended a tertiary care hospital in northeastern Mexico in 1997 and were followed up over a 5-year period. Molecular analysis included: 1) a mutation screening by heteroduplex analysis (HA) of BRCA1 and BRCA2 genes and 2) a sequence analysis.
RESULTS: Of 22 patients, 14 (63.6%) showed a variant band detected by heteroduplex analysis of the BRCA1 and BRCA2 genes: 8 polymorphisms, 4 mutations of uncertain significance, and 2 novel truncated protein mutations, one in BRCA1 (exon 11, 3587delT) and the other in the BRCA2 gene (exon 11, 2664InsA).
CONCLUSIONS: These findings support future studies to determine the significance and impact of the genetic factor in this Mexican women population.

Key words: breast cancer; BRCA genes; Mexico

OBJETIVO: Describir la presencia de mutaciones en los genes BRCA1 y BRCA2 y la evolución clínica de un grupo de mujeres con carcinoma mamario de inicio temprano (CMIT).
MATERIAL Y MÉTODOS: Se realizó un estudio hospitalario, prospectivo, en una muestra de 22 pacientes con CMIT (siete en etapa clínica IIA, ocho en la IIB y siete en etapa IIIA). Las pacientes fueron atendidas en un hospital del noreste de México en 1997 y se realizó un seguimiento clínico durante cinco años. El análisis molecular incluyó: 1) análisis heterodúplex (AH) para detectar bandas variantes en la secuencia de ADN de los genes BRCA1 y BRCA2, y 2) análisis de secuenciación.
RESULTADOS: De 22 pacientes, 14 (63.6%) mostraron banda variante por AH en los genes BRCA1 y BRCA2: ocho polimorfismos, cuatro mutaciones de significado incierto y dos mutaciones noveles con proteína truncada, una en BRCA1 (exón 11, 3587delT) y otra en BRCA2 (exón 11, 2664InsA).
CONCLUSIONES: Estos hallazgos apoyan el desarrollo de futuros estudios para determinar el impacto del factor genético en la población mexicana con CMIT.

Palabras clave: cáncer de mama; genes, BRCA; México

Breast cancer (BC) has evolved as one of the main causes of morbidity and mortality for women. Each year, at least a million women are diagnosed with this disease in the world and the number of deaths is estimated in 300 000.¹ Annually, 180 000 new cases and 30 000 deaths are reported in the United States.²

Mexico is a country with almost 98 million inhabitants; 51.1% are women.³ In 1993, the number of new BC cases registered was 5 739 and 20% of them were residing in the state of Nuevo León.⁴ In 1998, yearly cases increased to 9 490.⁴ The average BC mortality rate in Mexico is 10.45 /100 000 women. Nuevo Leon ranks third in mortality rate due to this cancer in the country, with 15.9/100 000 inhabitants, following by Mexico City and the state of Coahuila. The mortality rates due to BC are higher in the Northern states: Coahuila (16.1), Nuevo Leon, Sonora (14.5), Chihuahua (14.1), and Baja California (13.5).⁴

On the other hand, heredity is another important risk factor for BC. It is estimated that it contributes 3 to 10% to BC cases and up to 30% to early onset ones.⁶ Mutations in BRCA1 and BRCA2 genes are responsible for 90% of the inherited BC cases.⁷

Since there is a lack of information in Mexico about the contribution of BRCA1 and BRCA2 genes to BC, the aim of this case series, prospective, hospital-based study of 22 Mexican women with EOBC was to describe the clinical course of BC and the presence of mutations of the BRCA1 and BRCA2 genes.

Material and Methods

Our protocol was reviewed and approved by the Scientific and Ethics Committee of the Hospital of Specialities # 25, Mexican Institute of Social Security (IMSS). In our hospital an average of 250 new breast cancer cases are studied per year and 25 of them are women with EOBC. A sample of 22 consecutive women newly diagnosed with histologically confirmed EOBC, aged 35 years or younger at the time of diagnosis, was recruited from the hospital in 1997. The study design and objectives were explained to the patients who were asked to sign an informed consent. The clinical histories were obtained and followed up for 5 years. Also, each patient donated 15 ml of venous blood from which DNA was extracted.⁸ Mutations in the BRCA1 and BRCA2 genes were screened for by the heteroduplex analysis (HA). Variant bands observed in the HA were re-amplified and sequenced.⁹

Clinical and pathological data. Patients were interviewed by two of the authors (ALCG, PRF); a questionnaire was completed and patients' medical records were examined. Age at diagnosis and data about the clinical progress and outcome were also recorded. The tumor size was measured in centimeters at its largest diameter. All breast tumor slides were reviewed by one of the authors (ALCG). Tumors were classified histologically and recorded as ductal or lobular and subtypes of ductal carcinomas.¹⁰ The histological grade of differentiation of tumors was determined according to the modified Bloom-Richardson criteria.¹¹ In the mastectomy specimen, the total numbers of lymph nodes, as well as the number of specimens with metastasis were recorded. The sites of metastasis were also described. Patients attended the hospital at least four times during the first year following diagnosis, and twice each subsequent year.

Results

Clinical characteristics. Table I shows the more important clinical and tumoral findings. The identification number (ID) of patients is given in Tables II and III. All patients accepted to participate in the study and none were lost during 1997-2002. The age range was 24 to 35 years; 68% of patients completed elementary school and 82% were housewives. History of BC in the family was present in six out of 22 cases (27%) and two out of 22 (9%) patients had a first degree relative with BC. Of the two women with truncated protein mutations (TPM), the one involving the BRCA1 gene had only a paternal aunt with BC, while the other, who had the mutation in the BRCA2 gene, had a history of gastric and breast carcinoma in two paternal aunts; in the maternal line, she had two aunts with breast cancer, and one uncle with melanoma. Diabetes mellitus and arterial hypertension affected 9% of patients. Twenty patients had children (range 1-5) and the other two had not initiated active sexual life. Forty percent of patients with children breast-fed them. Eighty-six percent of patients did not smoke. According with the Bray classification, 36% of the patients had normal weight, 27% obesity grade I, 28% grade II, and 9% grade III.

Tumor histology. Twenty-one tumors were classified as ductal carcinomas and the remaining one as lobular in origin (Table I). Ductal carcinomas were poorly and moderately differentiated in 10 out of 22 and 11 out of 22 cases, respectively. The two women with TPMs had a classical invasive lobular carcinoma (BRCA1 gene mutation) and a medullar carcinoma (BRCA2 gene mutation). The medullar carcinoma showed non-caseous granulomas distributed within the tumor and around its border. Special stains for fungi, tuberculosis bacilli, and bacteria were negative.

DNA data. Table II shows a description of the mutations found for the BRCA1 and BRCA2 genes. Only 14 of the 22 patients showed a variant band when the mutations in the BRCA1 and BRCA2 genes were screened by the heteroduplex analysis. Thus, 64% of patients with EOBC had variations in the DNA sequence involving BRCA1 or BRCA2 genes. The molecular findings were: 8 patients had one or more polymorphisms, 4 women had mutations of uncertain significance (USM) and 2 other had 2 novel TPM, one in BRCA1 (exon 11, 3587delT) and the other in BRCA2 gene (exon11, 2664InsA). In general, exclusively BRCA2 variant bands were found in 71.4% of the cases, versus only 7.14% of BRCA1 variants; in 21.43%, patients had both BRCA1 and BRCA2 variant bands. Polymorphisms were more frequently found in the BRCA2 gene, especially in exon 27, followed by exons 11, 10 and 22. The TPM affected exon 11 in BRCA1 and BRCA2 genes.

Table III shows the clinical stage and molecular findings in BRCA1 and BRCA2 genes and Table IV summarizes the molecular findings according with the clinical stage.

All patients in the same clinical stage received the same treatment, including modified radical mastectomy, chemotherapy (5-fluoracile, epirubicine, cyclophosphamide) and radiotherapy. The five-year mortality among these women was about 45%. Table IV shows the number of dead women/total with the clinical stage at diagnosis (1/7 in IIA, 3/8 in IIB and 6/7 in IIIA). All deaths were caused by neoplastic activity and 80% occurred within the first 3 years following diagnosis. Fourteen patients developed metastatic disease, and among those, only 4 still remain alive.

Discussion

This follow-up study characterized 22 young women with EOBC who attended a tertiary care hospital in northeastern Mexico. Even though the results of this study cannot be extrapolated to the population at large, it provides useful information on which to base further studies.

Several risk factors particular to this group were observed. Obesity is a BC risk factor in postmenopausal women but apparently not in young women;¹² nevertheless, 14 out of 22 of our women had some degree of obesity. The prevalence of family history of BC in our patients was similar to those previously reported among Mexican women¹³ and similar to American and European studies.¹⁴ The prevalence of common diseases such as diabetes mellitus and arterial hypertension was similar to that reported in adults in the north of Mexico.¹⁵

With one exception, all fatalities had lymph node metastases (LNM) at diagnosis. The sole exception initially underwent partial lymph node resection, with only six non-metastatic lymph nodes dissected after careful examination of axillary fat; later, developing ipsilateral LNM and systemic dissemination. On the other hand, two patients initially presenting with LNM survived, one of whom remained asymptomatic at the end of the study, and the other, who had a poorly differentiated neoplasm, developed liver metastasis and had terminal disease. These outcomes are consistent with those reported in the relevant literature. In general, metastatic involvement of axillary lymph nodes is one of the most important prognostic factors for survival in BC. Without LNM, 5-year survival is about 80%, with one to three positive lymph nodes is 50% and only 21% with four or more nodes.¹⁶

In general, the frequency of mutations in BRCA1 and BRCA2 genes in our study sample seems to be very similar to that described in other studies of EOBC;^17-19 however, some of this mutations had not been described before (the two PTM and gly2793Arg in BRCA2 gene and Asp749Tyr in BRCA1 gene) and this can be explained by the genetic characteristic of our mestizo population.²⁰ If we consider only those mutations resulting in truncated proteins, BC in our patients was associated with mutations of BRCA1 and BRCA2 genes in 9 % of cases, which is also consistent with the percentage of cases in the literature attributable to these types of mutations in these genes.^{17, 18} Overall, depending on the population analyzed, the estimated percentage of mutations in young women with breast cancer is between 6 and 30%.¹⁹

As was mentioned before, the two truncating protein mutations detected in our Mexican women have not been reported previously in other countries.²¹ Since these women originated from northeastern Mexico, and had resided there for at least four generations, our observations may be identifying unique population mutations. In relation with the four cases with USM, further studies are needed to determine whether they correspond to clinically significant mutations or polymorphisms.

All the reported polymorphisms were in the BRCA2 gene, although some women also had them in the BRCA1 gene. It is well known that in different populations, the contribution of BRCA1 and BRCA2 genes may be different not only in polymorphisms but also in deleterious mutations; thus, in Jewish women, BRCA1 mutations are more frequent that BRCA2 ones, while in Italian women, the contribution of BRCA2 mutations predominates.²² The study of polymorphisms is important especially in a mestizo population, such as the Mexican case. Some of the polymorphisms described in these patients (CIVS7-34T in BRCA1 and A1093C in BRCA2 genes) have been previously described in Spain and elsewhere,²¹ including Africans (C8715+47T),²¹ thus denoting the influence of these origins.²⁰

Early studies of women with mutations in BC susceptibility genes did not show differences between sporadic and mutation-associated tumors.²³ More recent quantitative studies reveal higher nuclear grade, less positive expression to estrogen receptors, more pleomorphism and higher mitotic index in tumors associated with mutations than in sporadic tumors.²⁴ One of our two cases had a lobular carcinoma (BRCA1 mutation) and the other a medullar carcinoma (BRCA2 mutation). Both of these histological tumor types have a better prognosis than the common ductal adenocarcinoma. Furthermore, the observation of non-caseous granulomas in the tumor probably signals active anti-tumor cellular immunity, and more importantly, these patients were among the survivors and are now clinically asymptomatic.

Conclusions

Our data point to the need for a large population-based study to characterize the EOBC in Mexican women, in order to determine the type, frequency, and significance of variations in the sequences of BRCA1 and BRCA2 genes in Mexico.

Acknowledgments

To Professor RM Chandler- Burns† for his critical reading of our manuscript.

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Address reprint requests to
Prof. Dr. Hugo A Barrera-Saldaña
Departamento de Bioquímica
Facultad de Medicina de la Universidad Autónoma de Nuevo León
Avenida Madero y Dr. Eduardo Aguirre Pequeño
colonia Mitras Centro 64460, Monterrey, Nuevo León, México ]]> E-mail: hbarrera@fm.uanl.mx

Received on: November 7, 2003
Accepted on: February 1, 2005

We express our gratitude to the PAICYT Program, the Office of the President of UANL, the FOFOI: IMSS-2002/021/20111021 for funding, and Conacyt for the doctoral scholarship (93354) to ALCG. We also thank the PROMEP program of the Federal Secretariat of Education and the Mexican National Council of Science and Technology for support.