Keywords germline variants - genetic counseling - gynecological cancer risk - hereditary syndromes
Palavras-chave variantes de linhagem germinativa - aconselhamento genético - risco de câncer ginecológico
- síndromes hereditárias
Introduction
Breast cancer (BC) is the most common type of cancer in women, excluding non-melanoma
skin cancer. In 2020, there were almost 2.3 million new cases of BC worldwide, representing
24.5% of all cancer cases in women.[1 ] On the other hand, ovarian cancer (OC) is the most lethal gynecological malignancy.
In 2020, there were 313,959.414 OC cases and 207,252 deaths from it.[1 ] Endometrial cancer (EC) is the 6th most common type of cancer in women, with a worldwide incidence in 2020 of 417,367
and 97,370 deaths.[1 ] In Brazil, for the 2020 to 2022 biennium, the rate of new cases of OC, BC, and EC
is 6,650; 66,280 and 6,540 per year, respectively.[2 ] In Minas Gerais, 8,250 cases of BC, 630 of OC, and 670 of EC were estimated in 2020.[2 ]
Most gynecological cancers are sporadic, but ∼ 5% of EC, 25% of OC, and 10 to 30%
of BC have a hereditary pattern.[3 ]
[4 ] Most cases of hereditary BC and OC are attributable to mutations in one of the BRCA genes, which also increase the risk of other cancers.[3 ]
Hereditary tumor-associated syndromes such as Lynch syndrome (LS), Li-Fraumeni syndrome
(LFS), Peutz-Jeghers syndrome (PJS), and Cowden syndrome (CS) also represent an important
feature in the carcinogenesis of gynecological and breast tumors. Lynch syndrome is
an autosomal dominant inherited syndrome associated with a mutation in one or more
mismatch repair (MMR) pathway genes (MSH2, MLH1, MSH6, and PMS2 ) or in the EPCAM gene, which is MSH2 's regulator. About 15% of OC cases and 2 to 6% of EC cases are caused by LS.[5 ] Li-Fraumeni syndrome is associated with TP53 germline mutations, determining a high risk of developing primary cancers.[6 ] Peutz-Jeghers syndrome is a rare disorder associated with pathogenic mutations in
STK11 . This syndrome confers an elevated lifetime risk of several cancers as gastrointestinal,
BC and OC.[7 ] Cowden syndrome is a rare but clinically diagnosable multiple hamartoma syndrome,
and it is associated with PTEN germline mutations. Cowden syndrome confers up to 85% lifetime risk of BC and up
30% of EC.[8 ]
Other genetic variants also predispose to the neoplasm risk, such as PALB2 (high risk associated), and CHEK2, ATM, BARD1, and RAD51D (moderate risk associated).[9 ]
RAD51C, RAD51D, and BRIP1 mutations were also related to increased OC risk.[9 ]
The understanding of cancer genetic predisposition leads to better identification
of patients at risk; thus, physicians will be able to coordinate strategies for detection,
management, and prevention.[10 ] This study aims to evaluate the germline mutations profile in patients from different
regions of Minas Gerais state, who were submitted to genetic counseling for risk assessment
for BC, OC, and EC with possible hereditary patterns.
Methods
Between April 2017 and October 2018, a cohort of 382 patients undergoing genetic counseling
due to suspected hereditary cancer at a private genetic referral center in Belo Horizonte
was analyzed. In general, the main syndromes have gynecological cancer as part of
the also main phenotype, as explained in the introduction. This study was approved
by the Research Ethics Committee of Universidade Federal de Minas Gerais (CAEE: 01758418.0.0000.5149),
and prior written consent was accepted from all participants. Age, gender, naturality,
personal or family history of BC, OC, EC, and other cancers associated with hereditary
syndromes as well as genetics tests and their outcome were analyzed. Naturality was
defined according to the mesoregions of Minas Gerais, in agreement with the Brazilian
Institute of Geography and Statistics (IBGE). The criteria for genetic testing followed
the National Comprehensive Cancer Network (NCCN)[11 ] guideline for genetic/familial high-risk assessment, according to the time when
the study was performed, since NCCN guideline is actualized constantly. Thus, considering
the hereditary cancer predisposition syndromes, many other cancers were considered
as part of the family history phenotype. Although all patients received pretest genetic
counseling, the test methodology chosen varied according to the test availability
for each patient. Since it was a private service, most of the patients underwent genetic
tests following the approval of their current health plan or they paid for it on their
own. This means that some patients only had access to tests related to BRCA1 and BRCA2 genes, for example, and others have performed commercial panels for hereditary cancer.
The Human Genome Variation Society (HGVS) nomenclature guidelines (http://varnomen.hgvs.org/ ) were used to name the variants. The biological significance of variants reported
was assessed in the ClinVar (www.ncbi.nlm.nih.gov/clinvar/ ), Brazilian Genomic Variants (ABraOM - http://abraom.ib.usp.br/ ), dbSNP (https://www.ncbi.nlm.nih.gov/snp/ ), European Variant Archive (EVA - https://www.ebi.ac.uk/eva ), GeneCards (https://www.genecards.org/ ), GnomAD. (https://gnomad.broadinstitute.org/variant ), The Human Gene Mutation Database (http://www.hgmd.cf.ac.uk ), UniProt (https://www.uniprot.org/ ), Varsome (https://varsome.com/variant ), 1000 genomes (https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/ ), and BRCA Exchange (https://brcaexchange.org/ ) databases.
Results
Three hundred eighty-two patients with personal and/or family history of BC, OC, EC,
and other cancers associated with hereditary syndromes were selected at a referral
center in Belo Horizonte, Minas Gerais. Three hundred fifty-four patients were female
(354/382, 92.7%) and 28 were male (28/382, 7.3%). A total of 55.76% of patients (213/382)
were considered symptomatic, and 44.24% (169/382) were considered asymptomatic ([Fig. 1 ]).
Fig. 1 Reason for medical consultation. BC, personal history of breast cancer; FH CA, family
history of cancer without previously identified mutation; FamMut, identified family
mutation; non-gyn CA, personal history of non-gynecological cancer associated with
hereditary syndromes; OC, personal history of ovarian cancer; >1CA, personal history
of more than one type of cancer; EC, personal history of endometrial cancer.
Of the symptomatic patients, 159 had a personal history of BC, 10 of OC, 4 of EC,
32 were diagnosed with non-gynecological cancers associated with hereditary syndromes,
and 8 patients had more than one type of cancer: 1 had BC and OC associated, 1 BC
and EC, 1 BC, OC, and melanoma, and 5 had BC and non-gynecological cancers. Among
the asymptomatic patients, 54 sought genetic counseling due to previously identified
family mutation and 115 due to a family history of cancer without previously identified
mutation, and only 49.7% (84/169) were eligible for genetic tests. There were no patients
younger than 18 years (0/382), according to the exclusion criteria, 1 between 18 and
20 years (1/382, 0.26%), 45 between 21 and 30 years (45/382, 11.78%), 113 between
31 and 40 years (113/382, 29.58%), 101 between 41 and 50 years (101/382, 26.44%),
64 between 51 and 60 years (64/382, 16.75%), 43 aged 61 to 70 years (43/382, 11.26%),
8 aged over 70 years (8/382, 2.10%), and there were no data on the age of 7 patients
(7/382, 1.83%). Most patients were from Belo Horizonte Metropolitan Region (277/382),
followed by West of Minas Gerais, with 21/382 patients, 18/382 from the Rio Doce Valley,
10/382 from Campo das Vertentes, 9/382 from the North of Minas Gerais, 8/382 from
the Central Region, 8/382 from Zona da Mata, 4/382 from South/Southwest of Minas Gerais,
3/382 from Mucuri Valley, 2/382 from Triângulo Mineiro/Alto do Parnaíba, and 2/382
from Jequitinhonha. There were no cases from the Northwest of Minas Gerais. Three
cases had Ashkenazi ancestry, all from the Belo Horizonte Metropolitan Region, but
1 with family from Poland, 1 from Turkey, and 1 from Romania. Four cases were from
other countries: 3 from Lebanon and 1 from Italy. There were no data in the medical
records about the place of birth of 5 patients. A total of 85 variants were identified
in 72 patients. Among these, 50% had a personal history of BC (36/72), 1 of which
was associated with EC, and 1 associated with a non-gynecological cancer, 29 were
asymptomatic (29/72), 5 had previous history of non-gynecological cancers (5/72),
and 2 had a personal history of OC (2/72). Of the 29 asymptomatic patients, 23 were
patients with a family history of previously identified mutation and 6 had a family
history of cancer, with no previously identified mutation. Of the 15/72 patients with
variants in other genes, excluding BRCA1 /2, 5 were diagnosed with BC, 1 with BC and EC, 10 were asymptomatic or diagnosed
with non-gynecological cancer, but all had a family history of BC, and 1 asymptomatic
patient had a family history of OC. The age at diagnosis of BC in these patients ranged
from 33 to 64 years. Of these, there were 53 distinct mutations: 20 in BRCA2 , 18 in BRCA1 , 2 in APC , 2 in MUTYH , 2 in TSC1 , 2 in MSH2 , 1 in MLH1 , 1 in RAD51D , 1 in CHEK2 , 1 in PDGFRA , 1 in PTEN , and 1 in STK11 ([Fig. 2 ]) ([Table 1 ]).
Table 1
Different variants identified in the cohort study
P/LP
VUS
B/LB
APC del éxons 17–18 (1)
BRCA1 c.1713A > G (1)
APC c.5465T > A (1)
BRCA1 c.2037delinsCC (1)
BRCA1 c.220C > A (1)
BRCA1 c.804G > A (1)
BRCA1 c.211A > G (1)
BRCA2 c.1146A > T (1)
BRCA1 c.-19–115 T > C (2)
BRCA1 c.3331_3334delCAAG (2)
BRCA2 c.3196A > C (1)
BRCA1 c.2612C > T (1)
BRCA1 c.4675 + 1G > A (1)
BRCA2 c.5096A > G (1)
BRCA1 c.1486C > T (1)
BRCA1 c.4675 + 1G > T (6)
BRCA2 c.6988A > G (1)
BRCA2 c.7397 C > T (2)
BRCA1 c.470_471delCT (7)
BRCA2 c.8305G > C (1)
BRCA2 c.7806–14 T > C (2)
BRCA1 c.4964_4982del (1)
BRCA2 c.640G > A (1)
BRCA2 c.8755–66 T > C (2)
BRCA1 c.5266dupC (3)
CHEK2 c.319 + 3966G > A (1)
MUTYH c.1014G > C (1)
BRCA1 c.5467 + 3A > C (2)
PDGFRA c.718A > C (1)
STK11 c.1038C > T * (1)
BRCA1 c.798_799delTT (3)
RAD51C c.428A > G (1)
TSC1 c.625A > G (1)
BRCA1 del éxons 18–19 (1)
RAD51D c.26G > C (1)
BRCA1 c.4964C > T (1)
TSC1 c.3301G > A (1)
BRCA2 c.6591_6592del (4)
BRCA2 c.156_157insAlu (1)
BRCA2 c.1796_1800delCTTAT (2)
BRCA2 c.2978G > A (1)
BRCA2 c.2T > G (5)
BRCA2 c.4829_4830delTG (1)
BRCA2 c.5985delC (1)
BRCA2 c.6275_6276del (1)
BRCA2 c.6405_6409delCTTAA (2)
BRCA2 c.7819_7819delA (1)
BRCA2 c.9154C > T (3)
MLH1 del éxons 17, 18 e 19 (1)
MSH2 c.1894_1898del (1)
MSH2 c.2152C > T (1)
MUTYH c.536A > G (1)
PTEN c.264T > G (1)
Abbreviations: (#), number of probands; B/LB, benign/likely benign; P/LP, pathogenic/likely
pathogenic; VUS, variant of uncertain significance.
* This variant has 4 classifications VUS and 4 Benign. Variants in bold were identified
in more than one patient.
Fig. 2 Percentage of distinct variants detected in each gene evaluated.
The most frequent mutation was BRCA1 c.470_471delCT, which appeared in 7 cases (7/53) in 5 distinct families, followed
by BRCA1 c.4675 + 1G > T, with 6 cases (6/53), 5 of which were in the same family, and BRCA2 c.2T > G, with 5 cases (5/53) identified in 4 distinct families. Five variants (BRCA1 c.1713A > G, BRCA2 c.3196A > C, c.5096A > G, c.6988A > G, and RAD51C c.428A > G) are considered as VUS in ClinVar, and other tools were re-classified
as pathogenic in Varsome and the RAD51D c.26G > C was changed to benign variant. The following variants appear to have been
described for the first time in Brazil: APC c.5465T > A, BRCA1 c.1713A > G, c.220C > A, c.804G > A, c.5467 + 3A > C,; BRCA2 c.5985delC, c.7819_7819delA, c.6591_6592delTG, c.1146A > T, c.2978G > A, c.3196A > C,
c.6275_6276del, c.640G > A, c.8305G > C, CHEK2 c.319 + 3966G > A, MSH2 c.1894_1898del, PDGFRA c.718A > C, PTEN c.246T > G, RAD51C c.428A > G, TSC1 c.3301G > A, and TSC1 c.625A > G. Of the 72 patients who had mutations identified, the majority was from
the Belo Horizonte Metropolitan Region (38/72) followed by West of Minas Gerais, with
11/72 patients, 6/72 from the Rio Doce Valley, 4/72 from Zona da Mata, 3/72 from the
Central Region, 2/72 from the North of Minas Gerais, and, finally, South/Southwest
of Minas Gerais, Campo das Vertentes, Mucuri Valley, and Jequitinhonha with 1 patient
each ([Fig. 3 ]).
Fig. 3 Geographical distribution of variants identified in Minas Gerais State mesoregions,
according to the Brazilian Institute of Geography and Statistics (IBGE). Two patients
were from other countries, and one from another state. There were no patients from
the Northwest and Triângulo Mineiro regions.
There were no patients from the Northwest and Triângulo Mineiro regions. Two patients
were from other countries and one from another state. None of these patients declared
Ashkenazi ancestry.
Discussion
Genetic testing for patients with a high risk for gynecological cancer enables cancer
risk reduction strategies, such as salpingo-oophorectomy and mastectomy,[12 ] chemoprevention, and specific therapeutics, such as PARP inhibitors in BRCA-mutated
patients.[13 ] Besides, it allows differentiated cancer screening for early detection, such as
breast magnetic resonance image and mammography at an early age.[13 ] Among patients in the United States, for whom BRCA1 and BRCA2 mutation tests have become universal in clinical practice for OC patients, a reduction
of 40% is estimated in the incidence of OC and 37 to 64% of BC in 10 years in healthy
family members diagnosed with pathogenic mutation.[14 ]
It is necessary to identify the most common mutations in each population to develop
a specific panel, thus making the process more efficient and less costly. So, mutation
frequency studies should be conducted.[15 ] For the self-declared Ashkenazi patients, 3 founder mutations in BRCA (BRCA1 c.5266dupC, BRCA1 c.68_69del, and BRCA2 c.5946del) correspond to 98 to 99% of mutations identified.[16 ]
[17 ]
The Brazilian population is one of the most heterogeneous in the world.[17 ] Then, due to a lack of local studies, all recommendations are based on international
data. One of the major strengths of this study is that it is the first to evaluate
the germline mutations profile in the state of Minas Gerais.
The most frequent mutation found in this study was BRCA1 c.470_471delCT, which differs from those already performed in Brazil, in which BRCA1 c.5266dupC was the most common.[15 ]
[17 ] The BRCA1 c.470_471delCT mutation has been reported in 49 studies in the BRCA Exchange database,
and it is the most prevalent mutation in Hong Kong,[15 ] Malaysia, Southeast China,[18 ] Japan,[19 ] and Spain.[20 ]
BRCA1 c.470_471delCT was identified in 5 different families, 2 of which are native from
Vale do Rio Doce. One possible explanation is the beginning of commercial exploratory
activities between Japan and Brazil, in 1950, in this region. Another is a founder
mutation there. Of the 7 cases identified with this mutation, 5 had BC, with 3 of
them under the age of 40, and 1 had OC at 36 years old.
The most prevalent mutation in BRCA2 was c.2T > G, as previously described,[17 ]
[21 ] and it was present in 5 patients from 4 different families. Among these patients,
3 were diagnosed with BC, 2 of them younger than 40 years old. One patient diagnosed
at 33 years old had disease recurrence. In all cases, family history is significant
for BC and PC, which demonstrates the importance of the genetic test for predictive
medicine in gynecological oncology.
BRCA1 c.5266dupC is a founder mutation in Ashkenazi, and it is one of the most frequent
mutations worldwide, including in Brazil.[15 ] However, three cases were identified, none reported as Ashkenazi ancestry.
The Portuguese founder mutation BRCA2 c.156_157insAlu corresponds to more than a quarter of the BRCA1/2 mutations found in Portugal[20 ] and in Brazil, and it was frequently in Palmero's study.[17 ] Although Brazil received more than 2 million Portuguese between 1500 and 1991, this
variant had a low prevalence in other studies.[22 ] In our study, the mutation was found in only one patient. This mutation may not
have been identified in the tests performed due to low prevalence or because it is
a large insertion and may require a specific PCR-based test. However, all patients
are advised to keep their follow-up at the medical genetics service up to date and
review their clinical and laboratory data.
Mutations prevalent in other studies in Brazil, such as BRCA1 c.3331_3334delCAAG[17 ] and BRCA1 c.211A > G,[17 ] were found in 2 and 1 patients, respectively. In BRCA2 , the frequent variants BRCA2 c.2808_2811delACAA and BRCA2 c.5946ddelT, previously described,[17 ]
[21 ] were not identified in our cohort.
Another strength of our study is the evaluation of other genes involved in the predisposition
to gynecological cancers. In southern and southeastern Brazil, the founder mutation
TP53 c.1010G > A (p.R337H) was identified in 0.3% of the population,[23 ] which corresponds to 300 times more than any LFS-associated mutation.[24 ] Although a search for this specific mutation was requested for 11 (15.3%) patients
with suspected LFS or LFL in our cohort, none was identified as well as in one study
performed in Belo Horizonte.[25 ] This information makes us question what the real prevalence of this mutation in
Minas Gerais population is. Are the R337H carriers referred for genetic counseling?
And what is their real cancer risk?
We identified one patient diagnosed with a pathogenic mutation in APC without personal cancer history but with BC and colorectal cancer (CRC) family history.
Three patients were diagnosed with LS: one had EC at 54 years old and BC at 55 years
old, besides a family history of BC, CRC, pancreas cancer, and melanoma; one was asymptomatic
with OC, PC, CRC, and leukemia family history, and the other one had CRC at 51 years
old and has a family history of CRC and pancreas cancer.
The diagnosis of LS in patients and their families is extremely important, due to
the high risk of developing EC and OC and the evaluation of possible risk reduction
management. The 3 mutations identified here are pathogenic, two in MSH2 (MSH2 c.1894_1898del and MSH2 c.2152C > T) and 1 in MLH1 (MLH1 del exons 17, 18, and 19). The patient diagnosed with the pathogenic mutation MUTYH c.536A > G had no cancer personal history but had a family history of BC, CRC, PC,
and sarcoma. MUTYH mutations are associated with an elevated risk of CRC, EC, and BC.[26 ]
[27 ]
One patient with CS diagnostic and pathogenic mutation PTEN c.264T > G was also diagnosed with thyroid cancer at 24 years old. Although, she
has no family history of gynecological cancers, it is important to identify this mutation
due to the high risk of developing BC and EC.
In this study, 13 VUS were found: 2 in BRCA1 , 6 in BRCA2, and 1 in CHEK2 , PDGFRA , RAD51C , RAD51D, and TSC1 , each. Variants of undetermined significance is a gene sequence alteration with an
unknown consequence on the gene function.[28 ] Counseling patients with VUS results is challenging for at least two reasons. First,
it does not estimate the cancer risk and, therefore, does not allow guidance on preventative
measures. Secondly, the variant reclassification is a dynamic process and needs great
attention to patients' care. Initially, VUS must be treated as negative, and the risk
assessment should be based on family history.[29 ]
In this study, the patients were selected from a private genetic center, which limits
our study as most of the Brazilian population depends on the public health system.
Furthermore, genetic testing coverage by health plans determines the difference in
the methodology used between patients. Nevertheless, it allowed the evaluation of
a patient population undergoing genetic counseling for hereditary cancer within the
reality of clinical practice.
Conclusion
Considering the impact of a pathogenic/likely pathogenic mutation on the patient and
their family members, it is important to understand these population genetic profiles
to offer better genotype-phenotype correlation to guide clinical decisions and effective
management to reduce the cancer risk in a more democratic way which is adaptable to
health care.
