Germline molecular data in hereditary breast cancer in Brazil: Lessons from a large single-center analysis

Renata Lazari Sandoval; Ana Carolina Rathsam Leite; Daniel Meirelles Barbalho; Daniele Xavier Assad; Romualdo Barroso; Natalia Polidorio; Carlos Henrique dos Anjos; Andréa Discaciati de Miranda; Ana Carolina Salles de Mendonça Ferreira; Gustavo dos Santos Fernandes; Maria Isabel Achatz

doi:10.1371/journal.pone.0247363

. 2021 Feb 19;16(2):e0247363. doi: 10.1371/journal.pone.0247363

Germline molecular data in hereditary breast cancer in Brazil: Lessons from a large single-center analysis

Renata Lazari Sandoval ^1,^*,^#, Ana Carolina Rathsam Leite ^1,^#, Daniel Meirelles Barbalho ¹, Daniele Xavier Assad ¹, Romualdo Barroso ¹, Natalia Polidorio ¹, Carlos Henrique dos Anjos ¹, Andréa Discaciati de Miranda ², Ana Carolina Salles de Mendonça Ferreira ³, Gustavo dos Santos Fernandes ¹, Maria Isabel Achatz ⁴

Editor: Amanda Ewart Toland⁵

PMCID: PMC7895369 PMID: 33606809

Abstract

Brazil is the largest country in South America and the most genetically heterogeneous. The aim of the present study was to determine the prevalence of germline pathogenic variants (PVs) in Brazilian patients with breast cancer (BC) who underwent genetic counseling and genetic testing at a tertiary Oncology Center. We performed a retrospective analysis of the medical records of Brazilian patients with BC referred to genetic counseling and genetic testing between August 2017 and August 2019. A total of 224 unrelated patients were included in this study. Premenopausal women represented 68.7% of the cohort. The median age at BC diagnosis was 45 years. Multigene panel testing was performed in 219 patients, five patients performed single gene analysis or family variant testing. Forty-eight germline PVs distributed among 13 genes were detected in 20.5% of the patients (46/224). Eighty-five percent of the patients (91/224) fulfilled NCCN hereditary BC testing criteria. Among these patients, 23.5% harbored PVs (45/191). In the group of patients that did not meet NCCN criteria, PV detection rate was 3% (1/33). A total of 61% of the patients (28/46) harbored a PV in a high-penetrance BC gene: 19 (8.5%) BRCA1/2, 8 (3.5%) TP53, 1 (0.5%) PALB2. Moderate penetrance genes (ATM, CHEK2) represented 15.2% (7/46) of the positive results. PVs detection was statistically associated (p<0.05) with BC diagnosis before age 45, high-grade tumors, bilateral BC, history of multiple primary cancers, and family history of pancreatic cancer. According to the current hereditary cancer guidelines, 17.4% (39/224) of the patients had actionable variants. Nine percent of the patients (20/224) had actionable variants in non-BRCA genes, it represented 43.5% of the positive results and 51.2% of the actionable variants. Considering the observed prevalence of PVs in actionable genes beyond BRCA1/2 (9%, 20/224), multigene panel testing may offer an effective first-tier diagnostic approach in this population.

Introduction

Inherited germline pathogenic variants (PVs) in high or moderate penetrance breast cancer (BC) susceptibility genes are the underlying cause of approximately 15% of all BC cases [1, 2]. The implementation of preventive strategies may have an impact on cancer incidence and mortality in this high-risk population [3].

Several studies have demonstrated the cost-effectiveness of genetic testing, surveillance, prevention, and treatment strategies in PV carriers of cancer susceptibility genes [4]. Most studies are based on BRCA1/2 carriers. Other genes included in the multigene panels used for the investigation of hereditary BC still lack sufficient data on penetrance, genotype-phenotype correlations, as well as benefits of intensive surveillance and risk reduction surgeries related to a mortality reduction [5, 6].

The prevalence of germline mutations varies widely, depending on the selected studied population. In unselected populations, the detection rate of clinically actionable pathogenic variants is low [7]. Studies in populations with known founder mutations may detect mutations in 1.1–4.5% of individuals not selected based on a personal or family history of cancer [8]. Genetic testing based on family history approaches has a moderate to high diagnostic accuracy in predicting the detection of an inherited mutation, depending on the selected risk prediction tool [9]. Nevertheless, 50% of BRCA mutation carriers are missed through family history criteria [10].

Even though there are published data on hereditary BC among Latin American countries [11, 12], few studies have included non-BRCA genes and multigene panel testing [13, 14]. Brazil is the largest country in South America and is the most genetically heterogeneous [15]. Most studies performed in Brazilian cohorts on hereditary breast cancer have been performed in the Southern and Southeastern parts of the country [13, 16]. For this reason, epidemiological data about the prevalence of cancer predisposition syndromes in other parts of the country are needed.

Brasília is the capital of Brazil, located in the central region of the country, and was founded in 1960. A large number of internal migrants came from all over the country to build it and ended up populating the city. Therefore, Brasília residents represent a unique sample of the Brazilian population. To the best of our knowledge, no previous study has described BC germline data using genetic testing in this region. The purpose of this study was to describe the clinical data and frequency of germline PVs in Brazilian BC patients from Brasília.

Materials and methods

A total of 248 consecutive BC unrelated patients were referred for genetic counseling, between August 2017 and August 2019, at Hospital Sírio-Libanês (Brasília, Brazil), a tertiary oncology center. Ductal carcinoma in situ (DCIS) and invasive breast cancer were included in the study following NCCN criteria for further genetic evaluation. Twenty-four patients were excluded from the analysis because they did not undergo germline testing (Fig 1). A waiver of informed consent was approved by the Institutional Research Ethical Committee of the Hospital Sírio-Libanês (CAAE. 21735619.3.0000.5461). The Ethics Committee specifically reviewed and approved the protocol for our study.

Clinical information was retrospectively collected from the electronic medical records of patients. Electronic medical records were reviewed between November 2019 and January 2020. All personal and family history data were ascertained by institutional certified medical geneticists. All clinical and molecular data were de-identified before data sharing and analysis. The collected data included: age at cancer diagnosis; history of unilateral or bilateral breast cancer (synchronous or metachronous); histological subtype and tumor immunohistochemical profile; personal history of other primary cancers; family history of cancer (1^st, 2^nd, and 3^rd degree relatives); number of family members affected by BC; type of germline genetic test performed; number of analyzed genes, in case of multigene testing; and results of genetic testing, including PVs and variants of uncertain clinical significance (VUS). Detailed information about the gene panels studied in this cohort is described in the supplemental material.

All germline genetic tests were performed by commercial molecular diagnostic laboratories (S1 Table). Variants were classified according to the framework standardized by the American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) [17].

Statistical analyses

Continuous variables were tested for normality using the Kolmogorov-Smirnov and Shapiro-Wilk tests. Values are expressed as median and percentiles for non-parametric data, and as mean and standard deviation for parametric data. Categorical data are presented as absolute values and percentages and were tested using the Pearson χ2 test and Fisher’s exact test, when applicable.

Non-parametric data were compared using the Mann-Whitney U test for two independent samples or the Kruskal–Wallis test with a Müller-Dunn post-hoc test for three or more samples. Statistical significance was set at a p ≤ 0.05. Statistical analyses were performed using SPSS 21.0 IBM®.

Results

Study population

A total of 224 patients with BC were included in this study. Baseline and demographic characteristics of the cohort are described in Table 1. Four patients were male (1.8%, 4/224). Most patients were diagnosed with primary BC under the age of 50 years (66.1%, 148/224), with a median age at diagnosis of 45 years (95% Confidence interval [CI], 38–53). Thirty-nine patients (17.4%, 39/224) had more than one primary cancer, of whom 79.4% (31/39) had two primary cancers and 20.5% (8/39) had 3 or more primary cancers. BC was the primary cancer diagnosed in 208 out of 224 patients (92.8%). The remaining sixteen patients had previously been diagnosed with cancer, including thyroid cancer (6/16), melanoma (4/16), central nervous system (2/16), kidney (1/16), uterine cancer (1/16), lymphoma (1/16), and pancreatic cancer (1/16).

Table 1. Baseline and demographic characteristics.

Characteristics	Patients with no germline pathogenic variants, n = 178 (%)	Patients with germline pathogenic variants, n = 46 (%)	P = value
Total cohort = 224	Patients with no germline pathogenic variants, n = 178 (%)	Patients with germline pathogenic variants, n = 46 (%)	P = value
Gender
Male	1 (0.6)	3 (6.5)	.007^**
Female	177 (99.4)	43 (93.5)
Number of primary cancers
1	151 (84.8)	34 (73.9)	.046^**
2	22 (12.4)	9 (19.6)
≥ 3	5 (2.8)	3 (6.5)
Age at breast cancer diagnosis
≤ 31 years	11 (6.2)	7 (15.2)	.009^**
32–44 years	68 (38.2)	21 (45.7)
45–49 years	36 (20.2)	1 (2.2)
≥ 50 years	63 (35.4)	17 (37)
Menopausal status
Premenopausal	113 (67.3)	32 (74.4)	.366
Postmenopausal	55 (32.7)	11 (25.6)	.366
Missing data	10	3
Laterality
Unilateral	171 (96.1)	40 (87)	.018^**
Bilateral	7 (3.9)	6 (13)	.018^**
Tumor histology
NTS	118 (74.7)	36 (87.8)	.345
DCIS	25 (15.8)	4 (9.8)
ILC	13 (8.2)	1 (2.4)
Others^*	2 (1.1)	0 (0)
Missing data	20	5
Tumor Grade
G3	48 (36.6)	20 (64.5)	.002^**
G2	76 (58.0)	7 (22.6)
G1	7 (5.3)	4 (12.9)
Missing data	47	15
Immunohistochemical profile
HR+ HER2-	81 (54.4%)	17 (48.6)	.384
HR+ HER2+	29 (19.5)	8 (22.9)
HR- HER2+	14 (9.4)	1 (2.9)
Triple negative	25 (16.8)	9 (25.7)
Missing data	29	11
Family history of cancer
Negative or unknown	20 (11.2)	3 (6.5)	.585
Positive	158 (88.8)	43 (93.5)	.585
Breast	99 (55.6)	30 (65.2)	.215
Ovarian	12 (6.7)	5 (10.9)	.681
Pancreatic	17 (9.5)	11 (23.9)	.048^**
Prostate	46 (25.8)	16 (34.8)	.441

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Abbreviations: NTS- invasive carcinoma of no special type; DCIS, ductal carcinoma in situ; ILC, invasive lobular carcinoma; IHC, immunohistochemistry; G, grade; HR, hormonal receptors; HER2, human epidermal growth factor receptor 2.

*Others: sarcomatous cancer and malignant phyllodes.

**Statistically significant.

Premenopausal BC was present in 68.7% of patients (145/211). Thirteen patients (5.8%, 13/224) had bilateral cancer, including 4 synchronous and 9 metachronous cancers. Invasive carcinoma of no special type (NTS) was present in 77.4% (154/199) of the patients, followed by ductal carcinoma in situ (DCIS) [14.6% (29/199)], invasive lobular carcinoma (ILC) [7% (14/199)], sarcomatous cancer or malignant phyllodes [1% (2/199)]. High-grade tumors (grade 3) represented 42% (68/162) of the cases, grade 2 51.2%, and grade 1 6.8%. Sixty-two cases had no description of tumor grade in the records. According to hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) status, HR+/HER2- tumors represented 53.3% of the cases (98/184), HR+/HER2+ 20.1% (37/184), HER2-enriched (HR-/HER2+) 8.1% (15/184), and 18.5% of cases were triple negative (HR-/HER2-). The immunohistochemical profile of forty cases was not available in the medical records.

Considering the family history of cancer, 89.7% (201/224) of the patients had, at least, one first- or second-degree family member affected by cancer. Sixty-four percent (129/201) of patients referred having family members with BC. Ovarian, pancreatic, and prostate cancer were present in 8.5% (17/201), 13.9% (28/201), and 30.9% (62/201) of family relatives, respectively. Seventy-one patients (31.7%) reported to have two or more first- or second-degree relatives with BC. Twenty-three patients (10.2%) did not provide any information about family history or had no known relatives with cancer.

Frequency and spectrum of pathogenic germline variants

A total of 219 patients (97.7%) underwent multigene panel testing, 3 patients (1.3%) had family variant testing, and 2 patients (0.8%) underwent only BRCA1/2 sequencing. Ten patients underwent multigene panel testing, including up to 50 genes, and 206 patients underwent gene testing with panels having 80 or more genes (S1 Table). The number of genes evaluated in the panel was not available for three patients. The number of tested genes varied according to the health insurance approval and the options of patients after pretest genetic counseling.

Forty-eight PVs were detected in 46 patients (20.5%, 46/224) (Fig 2). More details of the PVs detected are described in Table 2 and S2 Table. Two patients had more than one PV detected: PV in BRCA2 and a monoallelic PV in MUTYH, PV in BRCA1 and a monoallelic PV in CTC1.

Table 2. Pathogenic and likely pathogenic variants detected.

Gene	Variant	Classification	dbSNP or Variation ID	Number of patients
ATM	c.7913G>A (p.Trp2638*)	PV	rs377349459	1
BARD1	c.176_177del (p.Glu59Alafs*8)	PV	rs1057517589	2
BRCA1	del exons 8–19	PV	Variation ID: 126018	1
BRCA1	c.132C>G (p.Cys44Trp)	LP	rs876658362	1
BRCA1	c.441+2T>A (splice donor)	PV	rs397509173	1
BRCA1	c.791_794del (p.Ser264Metfs*33)	PV	rs80357707	1
BRCA1	c.850C>T (p.Gln284Ter)	PV	rs397509330	1
BRCA1	c.1115G>A (p.Trp372*)	PV	rs397508838	1
BRCA1	c.1687C>T (p.Gln563*)	PV	rs80356898	2
BRCA1	c.3598C>T (p.Gln1200*)	PV	rs62625307	1
BRCA1	c.5177_5180delGAAA (p.Arg1726Lysfs*3)		rs80357867	1
BRCA1	c.5266dupC (p.Gln1756Profs*74)	PV	rs80357906	3
BRCA2	c.156_157insAlu (p.Lys53Alafs)	PV	Variation ID: 126018	1
BRCA2	c.1310_1313del (p.Lys437Ilefs*22)	PV	rs80359277	1
BRCA2	c.3680_3681del (p.Leu1227Glnfs*5)	PV	rs80359395	1
BRCA2	c.2512A>T (p.Lys838*)	PV	rs747578057	1
BRCA2	c.5073dupA (p.Trp1692Metfs*3)	PV	rs80359479	1
BRCA2	c.6405_6409del (p.Asn2135Lysfs*3)	PV	rs80359584	1
CHEK2	c.319+2T>A (splice donor)	LP	rs587782401	1
CHEK2	c.349A>G (p.Arg117Gly)	LP	rs28909982	2
CHEK2	c.593-1G>T (splice acceptor)	LP	rs786203229	1
CHEK2	c.846+1G>C (splice donor)	LP	rs864622149	1
CHEK2	c.1008+2T>G (splice donor)	LP	rs1555915295	1
MUTYH	c.305-1G>C (splice acceptor)	PV	rs372267274	1
MUTYH	c.933+3A>C (Intronic)	PV	rs587780751	1
MUTYH	c.1187G>A (p.Gly396Asp)	PV	rs36053993	2
MSH6	c.1519dupA (p.Arg507Lysfs*8)	PV	rs876658881	1
PALB2	Deletion exon 2–3	PV	-	1
RAD51C	c.709C>T (p.Arg237*)	PV	rs770637624	2
RAD51D	c.694C>T (p.Arg232*)	PV	rs587780104	1
RECQL4	c.1166_1167del (p.Cys389Phefs*33)	PV	rs34134064	1
TP53	Partial deletion exon 5	PV	-	1
TP53	c.733G>A (p.Gly245Ser)	PV	rs28934575	1
TP53	c.1010 G>A (p.Arg337His)	PV	rs121912664	6

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Abbreviations: PV, Pathogenic variant; LP, Likely pathogenic.

A total of 191 patients (85.3%, 191/224) fulfilled NCCN hereditary BC testing criteria. Among these patients, 23.5% harbored PVs (45/191). In the group of patients that did not meet NCCN criteria, PV detection rate was 3% (1/33) (S3 Table).

PVs were distributed among 13 genes. Sixty-one percent of patients (28/46) had PVs in a high-penetrance gene for BC: 19 (8.5%) in BRCA1/2, 8 (3.5%) in TP53, and 1 (0.5%) in PALB2. Moderate penetrance genes for BC (ATM, CHEK2) represented 15.2% (7/46) of the positive results: 6 (13%) patients had PV in CHEK2 and 1 (2.1%) in ATM. PVs in genes considered to have a potential increased risk of BC (BARD1, RAD51C, RAD51D) or an unknown risk/insufficient data (MUTYH, MSH6, RECQL4) were found in 23.9% (11/46) of patients. According to current guidelines [18], 39 of the 224 tested patients (17.4%) had actionable variants. Twenty out of 39 (51.2%) actionable variants were found in non-BRCA genes.

Correlation between test positivity, germline genotype, and clinical data

The diagnosis of bilateral BC occurred in 13% (6/46) of patients who harbored a germline PV (2 BRCA2 carriers, 2 CHEK2, 1 BARD1, and 1 RAD51C). Bilateral BC was predictive of a positive test (p = 0.018). Considering all the cases of bilateral BC (13/224), 31% (4/13) harbored PVs in non-BRCA genes. Two patients had ipsilateral breast tumors; however, it was not possible to determine whether it was a new primary tumor or a local recurrence. Both had a negative test result.

The majority of PVs were nonsense or frameshift (24/48, 50%). There were 14 frameshift variants (29.2%), 10 nonsense variants (20.8%), 13 missense variants (27%) and 7 splice site variants (14.6%). The remaining four variants (4/48, 8.4%) were pathogenic copy number variations (CNVs), including the diagnosis of one Alu insertion in BRCA2 (c.156_157insAlu, a Portuguese founder mutation), one case of BRCA1 exons 8–19 deletion, one PALB2 exon 2–3 deletion, and one TP53 partial deletion of exon 5 (possibly mosaic). The partial deletion in TP53, possibly in mosaic, was not confirmed using fibroblast genetic testing due to patient death.

A young age at BC diagnosis (< 45 years) was statistically associated with PV detection (p = 0.040). Most of the patients with a positive test result had a cancer diagnosis before the age of 50 years (63%, 29/46). It included 45.6% of patients aged 32–44 years (21/46), 15.2% (7/46) who were under 31 years old, and 2.2% (1/46) aged 45–49 years. High grade tumors were associated with a higher probability of PV detection (p = 0.002). Although premenopausal BC alone was not statistically associated with PV detection, 62% of premenopausal women with high-grade BC had a PV identified in the genetic test (p = 0.008).

Multiple primary tumors were more common among patients with a positive test compared to patients with a negative/VUS test result (26.1% vs. 15.2%, p = 0.042). Multiple family members affected by breast, prostate, or pancreatic cancer were associated with a higher probability of PV detection (p = 0.010). There was a statistical association between PVs and a family history of ovarian cancer only for PVs in the BRCA1 gene (p < 0.001).

At least one VUS was described in the genetic test reports of 140 patients (62.5%) (S4 Table). Most patients had only 1 VUS detected (82/140; 58,6%), but in approximately 41,4% (58/140) of cases, 2–6 VUS were described. Excluding patients with VUS and confirmed PV (33/140), VUSs were described in 107 patients. The frequency of VUS increased according to the number of genes included in the genetic test (p = 0.006).

Discussion

Hereditary BC has significant genetic heterogeneity. The National Comprehensive Cancer Network (NCCN) guidelines (version 1.2021) recommend genetic evaluation, including the genes BRCA1, BRCA2, TP53, ATM, CDH1, CHEK2, NBN, NF1, and PALB2 for high-risk breast and/or ovarian cancer patients [18]. There is cumulative evidence that variants in BARD1, BRIP1, MSH2, MLH1, MSH6, PMS2, RAD51C, and RAD51D may also be implicated in hereditary BC [19–22]. Next-generation sequencing (NGS) technologies have been an effective method in a multigene testing scenario [23]. In addition, the costs of DNA sequencing have been decreasing significantly with the use of NGS [24]. Notwithstanding, there are global disparities in genetic testing accessibility.

Genetic testing is not accessible for the majority of the population from developing and underdeveloped countries. Although there is an international current debate about BC genetic testing based on clinical criteria versus BC universal testing [25–27], Brazil and other Latin America countries face limited access. The main reported barriers are related to the lack of structured genetic counseling and genetic testing networks, insufficient number of trained professionals in high risk cancer assessment, absence of genetic testing access in the public health system, limited health insurance coverage, costs of genetic testing and lack of national policies [28, 29].

Recent data, collected in 2020 by the Brazilian National Agency of Supplementary Health, estimated that only 24% of the Brazilian population has access to health insurance [30]. Therefore, more than 163 million people depend on the Brazilian national public health system, which has no access to genetic tests for the investigation of cancer predisposition syndromes. Since 2018, supplementary health care must cover germline genetic testing, according to some clinical criteria (much more restricted than those described in NCCN guidelines). The present study included patients from supplementary health care and, for this reason, 90% of the initial sample had access to genetic testing. A total of 17.4% of the tested patients (39/224) had actionable variants according to the current NCCN guidelines [18].

The detection rate of actionable PVs varies widely depending on the selected studied population and the genetic testing approach (founder mutations, single gene analysis, copy number variation evaluation, multigene panel testing). The present cohort consisted of consecutive BC patients referred to genetic counseling selected due to the suspicion of hereditary BC. The overall detection rate of PVs was 20.5% (46/224). According to NCCN criteria, 85.3% (191/224) of our cohort met hereditary BC testing criteria, among these patients 23.6% (45/191) harbored PVs. Whereas, among 33 patients that did not met NCCN criteria, only one harbored a PV (3%). The detection rate of PVs was similar to other multigene panel testing studies based on hereditary breast and ovarian cancer (HBOC) criteria [5, 13, 14, 31–33].

Fifty-seven percent of the patients with a positive test result harbored PVs in high-penetrance BC genes (41.3% in BRCA1/2, 17.4% in TP53, and 2.2% in PALB2), 15.2% in moderate penetrance BC genes (2.2% in ATM and 13% in CHEK2), and 23.9% in genes considered to be associated with a BC potential increased risk or an unknown risk. Nine percent of the patients (20/224) had actionable variants in non-BRCA genes, representing 43.5% of the positive results and 51.2% of the actionable variants.

The detection rate of PVs in moderate penetrance BC genes varies from 2 to 8% [5, 33–35]. Our cohort was enriched by PVs in moderate penetrance BC genes (15.2% of positive results, 9% of overall genetic tests). Another Brazilian study with individuals from the Northeast of the country also found a high prevalence of PVs (32% of positive results) in moderate penetrance BC genes (12). These findings suggest that Brazilian patients should have access to multigene panel testing.

Although there is a higher frequency of CHEK2 founder mutations (c.1100delC, c.470T>C) in European ancestry populations, these mutations were not observed in our cohort. Other Brazilian studies have also showed no enrichment of CHEK2 European founder mutations in BC Brazilian patients [36–39]. The majority of CHEK2 PVs reported in our study affected RNA splicing (Table 2).

There are limited studies from Brazil and Latin American countries with BC germline characterization by multigene panel testing. A Brazilian BC study from the Northeast of the country showed a PV frequency of 17% (27/157), 68% (13/19) harbored a PV in BRCA1/2 genes and 32% (6/19) in moderate penetrance BC genes (12). Most of these patients were tested using a 33-gene panel. A research group from the Brazilian Southeast region performed a 21-gene panel in 95 women with a personal history of BC or HBOC clinical suspicion based on family history criteria [14]. Twenty-three percent of the patients harbored a PV in BRCA1/2 and TP53 genes. Eighty-five women from Colombia, meeting HBOC criteria, had germline testing with a commercial 25-gene hereditary cancer panel [33]. Twenty-two percent of the patients (19/85) harbored a PV in a cancer susceptibility gene.

NGS approaches may have limited ability to detect copy number variations (CNVs). Supplemental methods, along with NGS analysis, are required to validate CNV detection from NGS panels [40]. In the present study, 8.7% of the patients with PV (4/46) had pathogenic CNVs detected using multigene panel testing: one Alu insertion in BRCA2 (c.156_157insAlu), one BRCA1 exon 8–19 deletion, one PALB2 exon 2–3 deletion, and one TP53 partial deletion of exon 5. Previously, Ewald et al. (2016) observed a 3.4% prevalence of BRCA1/2 CNVs among 145 unrelated Brazilian individuals at risk of HBOC syndrome, which included three cases of Alu insertion in BRCA2 (c.156_157insAlu) [41].

The prevalence of Li-Fraumeni syndrome (LFS) in our cohort was 3.5% (8/224). Giacomazzi et al. (2014) reported a 3.4% prevalence of p.R337H in Brazilian women diagnosed with BC who met the criteria for HBOC [42]. Considering all the patients with a detected PV in the present cohort, LFS represented 17.4% (8/46) of the PV carriers. Six out of eight patients diagnosed with LFS in this study carried the Brazilian TP53 p.R337H variant, which corresponds to a prevalence of 2.7% (6/224). Similarly, Hahn et al. (2018) observed a frequency of the p.R337H variant in 2.5% (6/239) of Brazilian patients with BC diagnosed before age 46, unselected by family history [43]. It is well known that LFS has a higher prevalence in Brazil due to the TP53 founder mutation c.1010G>A (p.Arg337His), also known as p.R337H [44]. These findings raise the question of whether this prevalence justifies routine screening of all Brazilian women with BC, despite the Chompret criteria. In the Ashkenazi Jewish population, in which BRCA1/2 founder mutations are present in 2.5% of the individuals, cost-effectiveness studies have implied that population testing is justified [45].

Patients with cancer predisposition syndromes have a higher risk of developing a second primary BC, especially for BRCA 1/2 mutation carriers [46]. Nevertheless, 8–36% of patients with bilateral BC harbor PVs in other genes beyond BRCA [32, 47, 48]. Bilateral BC represented 5.8% (13/224) of our cohort. Thirty-one percent (4/13) of patients with bilateral BC harbored PVs in non-BRCA genes (CHEK2, BARD1 and RAD51C). These results are in concordance with previous studies [32, 47].

The concepts of clinical validity and clinical utility are important for the implementation of multi-gene testing and the development of guidelines for hereditary BC [6]. Genes classified as BC potential increased risk or unknown risk are not expected to change BC screening or management; nevertheless, they may provide information for high-risk assessment or risk reduction surgeries for other cancer sites. Four out of 46 carriers of PVs (8.7%) harbored a PV in RAD51C, RAD51D, and MSH6. RAD51C and RAD51D confer higher risks of ovarian cancer and MSH6, for endometrial, ovarian, and colorectal cancer. Monoallelic MUTYH PVs were not included as actionable in the current study, although colonoscopy is recommended at 40 years, if there is a family history of colorectal cancer. Despite the fact that monoallelic MUTYH PV is not an uncommon finding in BC patients undergoing multigene testing, it is not associated with BC risk [49, 50].

This study had several limitations. It was retrospective and had a limited sample size compared to multigene testing studies around the world. However, it represents the largest Brazilian BC cohort from a single institution tested using a multigene panel for hereditary BC [13, 14]. It is also the first BC germline characterization from the Center-West of the country. These findings require validation in other cohorts. The study was conducted at a private cancer center and, for this reason, the assessed population may differ from that of the general community. Furthermore, it consisted of a high-risk population for hereditary cancer with a median age of 45 years at BC diagnosis, mostly composed of patients with premenopausal BC (68.7%), a positive family history for cancer (89.7%), and a personal history of multiple primary cancers (17.4%).

Conclusion

This is the first study with germline molecular data from patients affected by BC in the Center-West of Brazil. We found a 20.5% prevalence of PVs. Seventeen percent of these patients had actionable variants according to the current guidelines. Nine percent of the patients had actionable variants in non-BRCA genes, representing 43.5% of the positive results and 51.2% of the actionable variants. Eighty-five of the patients fulfilled NCCN hereditary BC testing criteria, among these patients 23.5% harbored PVs. In our study, BC prior to 45 years, multiple primary cancers, high-grade tumors, bilateral BC, and a family history of pancreatic cancer were features associated with a higher probability of PV detection. Multigene panel testing may offer an effective first-tier diagnostic approach in this high-risk population.

Supporting information

S1 Table. Characteristics of genetic tests performed.

(DOCX)

Click here for additional data file.^{(13.5KB, docx)}

S2 Table. Clinical characteristics of patients with pathogenic and likely pathogenic variants.

(DOCX)

Click here for additional data file.^{(23.9KB, docx)}

S3 Table. Number of patients who meet current genetic testing criteria according to the genetic test result.

(DOCX)

Click here for additional data file.^{(12.9KB, docx)}

S4 Table. Variants of uncertain significance detected.

(DOCX)

Click here for additional data file.^{(23.7KB, docx)}

Data Availability

All relevant data are within the manuscript and its Supporting Information files. The original data is available in an excel file.

Funding Statement

The authors received no specific funding for this work.

References

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PLoS One. doi: 10.1371/journal.pone.0247363.r001

Decision Letter 0

Amanda Ewart Toland

9 Nov 2020

PONE-D-20-32343

Germline molecular data in hereditary breast cancer in Brazil: lessons from a large single-center analysis.

PLOS ONE

Dear Dr. Sandoval,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

1. It is unusual that missense variants make up the majority of pathogenic variants (unless we are considering TP53). More details should be included on how variants were classified. Furthermore, a table of all pathogenic and VUS found in the paper should be included in the supplemental materials.

2. It is not clear why DCIS is considered as a breast cancer diagnosis. More rationale for this should be included or consider only using primary invasive breast cancer as a diagnosis.

3. On Figure 2, the "no PV or VUS" group should be renamed as Likely benign or no variants. Figure 2 might be improved by using color.

4. In the Discussion expand discussion of the other Brazilian studies that have been published and how this study compares. Also include any additional studies of other nearby countries.

5. Deposit sequence variants found into a publicly accessible database.

Please submit your revised manuscript by Dec 24 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: October 25th, 2020

Comments to authors:

General:

The MS “Germline molecular data in hereditary breast cancer in Brazil: lessons from a large single-center analysis” by Renata Lazari Sandoval, M.D., M.Sc et al, describes the results using a panel of genes analyzed by NGS in breast cancers patients, concluding the benefits for use of panel genes. This MS has interesting regional results, although, as disclosed by the authors, the number of cases listed, nowadays, are a clear limitation if a strong conclusion should be draft.

A few comments to be considered:

a) Supplementary material table, is difficult to follow since it is based on the patient ID rather than in a field related to the topic of the MS (i.e. pathogenic variant, or IHC o whatever is chosen)

b) In Figure 2. a straight category might be more adequate (like “Benign”) rather that “No PV or VUS”

c) There are representative studies in Brazil that were ignored in this MS, and the utility is obvious to contrasting the results of the cohorts. Although the authors disclosed:

This study had several limitations. It was retrospective and had a limited sample size. However, it represents the largest Brazilian BC cohort from a single institution tested using a multigene panel for hereditary BC (12,13). These findings require validation in other cohorts. The study was conducted at a private cancer center and, for this reason, the assessed population may differ from that of the general community. Furthermore, it consisted of a high-risk population for hereditary cancer with a median age of 45 years at BC diagnosis, mostly composed of patients with premenopausal BC (68.7%), a positive family history for cancer (89.7%), and a personal history of multiple primary cancers (17.4%).

It might be a good improvement to discuss comparing with larger cohorts, even of other regions, from the large country Brazil.

d) Discussing the application of the NCCN guidelines upon the findings in the present work, may be interesting

e) A good idea is to deposit the variants in a public database for the free access to the worldwide scientific community

Reviewer #2: Methods: I would recommend using primary breast cancer as an inclusion criterion.

Results:

Study population: I would recommend excluding the remaining sixteen patients who had previously been diagnosed with other cancer because according to the title this paper is focused on breast cancer.

Table 1: I would recommend including an extra column with the information of the total cohort for each item.

Frequency and Spectrum of Pathogenic Germline Variants: I recommend including in supplementary data analyzed genes in each group of patients. I would also recommend presenting in the article text a table with the PV detected.

Correlation between test positivity, germline genotype, and clinical data: I recommend including a table with the VUS detected in supplementary data.

**********

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Reviewer #1: No

Reviewer #2: Yes: Laura Cifuentes-C

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PLoS One. 2021 Feb 19;16(2):e0247363. doi: 10.1371/journal.pone.0247363.r002

Author response to Decision Letter 0

25 Dec 2020

Manuscript PONE-D-20-32343

Response to reviewers

Dear Dr. Toland,

Thank you for giving us the opportunity to submit a revised version of the manuscript “Germline molecular data in hereditary cancer in Brazil: lessons from a large single-center analysis”. We are grateful for the time and effort that you and the reviewers dedicated to providing feedback on our manuscript. All the comments provided the possibility of relevant improvements to our manuscript.

We have incorporated most of the suggestions made by the reviewers and clarified all the points raised during the review process. Authors´ responses are described in blue in a point-by-point response to the reviewers’ comments and concerns. In the first section the editor´s points were addressed, in the second section Reviewer 1 and in the third section Reviewer 2.

Changes performed in the revised manuscript are highlighted in yellow. Page numbers refer to the revised manuscript file with tracked changes.

Section 1- Editor´s comments to the authors:

1. It is unusual that missense variants make up the majority of pathogenic variants (unless we are considering TP53).

Author response: We revised all the 48 likely pathogenic/pathogenic variants observed in this study: 10 nonsense, 14 frameshift, 4 copy number variants, 13 missense, 7 splice site. We thank you for the opportunity to correct this error, missense variants did not make up the majority of pathogenic variants, it represented 27% of pathogenic variants (13/48). Nonsense and frameshift variants, together, represented the majority of pathogenic variants (24/48, 50%).

Changes in the text of the revised manuscript: page 11 line 160, “The majority of PVs were nonsense or frameshift (24/48, 50%). There were 14 frameshift variants (29.2%), 10 nonsense variants (20.8%), 13 missense variants (27%) and 7 splice site variants (14.6%). The remaining four variants (4/48, 8.4%) were pathogenic copy number variations (CNVs), including the diagnosis of one Alu insertion in BRCA2 (c.156_157insAlu, a Portuguese founder mutation), one case of BRCA1 exons 8-19 deletion, one PALB2 exon 2-3 deletion, and one TP53 partial deletion of exon 5 (possibly mosaic). The partial deletion in TP53, possibly in mosaic, was not confirmed using fibroblast genetic testing due to patient death.”

More details should be included on how variants were classified. Furthermore, a table of all pathogenic and VUS found in the paper should be included in the supplemental materials.

Author response: All germline genetic tests were performed by commercial molecular diagnostic laboratories. We created the table S1 (supplementary material) with the characteristics of the genetic tests performed. Variant classification performed by each commercial laboratory was specified.

Variants were classified according to the framework standardized by the American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP). The majority of the genetic tests (196/219, 90%) were performed in the laboratory Invitae (San Francisco, California, USA). The Invitae Clinical Genomic Group also use the Sherloc framework for variant classification besides the ACMG-AMP variant classification guidelines.

All likely pathogenic/pathogenic were described in Table 2 in the manuscript. More detailed information about each patient that harbored a PV was described in S2 (supplementary material). All VUS were described in S4 (supplementary material). A column in Table 2 and S4 describes the variant identification as rsID (The Single Nucleotide Polymorphism Database identification, a free public archive for genetic variation) or Clinvar variant identifier number (variation ID).

Changes in the the text of the revised manuscript: page 4 line 74, “All germline genetic tests were performed by commercial molecular diagnostic laboratories (S1 Table). Variants were classified according to the framework standardized by the American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) (16).”

2. It is not clear why DCIS is considered as a breast cancer diagnosis. More rationale for this should be included or consider only using primary invasive breast cancer as a diagnosis.

Author response: Although DCIS is a non-invasive or pre-invasive breast cancer, the National Comprehensive Cancer Network guidelines for Genetic/Familial High-Risk Assessment (https://www.nccn.org/professionals/physician_gls/default.aspx) include both invasive and ductal carcinoma in situ breast cancers as a criteria for further genetic evaluation (footnote d, version 1.2021, page 17 from NCCN guideline). Some studies also have showed that DCIS should be a criteria for hereditary breast cancer risk assessment as it seems to occur equally in sporadic and hereditary breast cancer cases. In mutation carriers it seems to occur at an earlier age.

References:

Hwang ES, McLennan JL, Moore DH, Crawford BB, Esserman LJ, Ziegler JL. Ductal carcinoma in situ in BRCA mutation carriers. J Clin Oncol. 2007 Feb 20;25(6):642-7. doi: 10.1200/JCO.2005.04.0345.

Bayraktar S, Elsayegh N, Gutierrez Barrera AM, et al. Predictive factors for BRCA1/BRCA2 mutations in women with ductal carcinoma in situ. Cancer. 2012 Mar 15;118(6):1515-22. doi: 10.1002/cncr.26428.

Liu Y, Ide Y, Inuzuka M, et al. BRCA1/BRCA2 mutations in Japanese women with ductal carcinoma in situ. Mol Genet Genomic Med. 2019 Mar;7(3):e493. doi: 10.1002/mgg3.493.

Changes in the text of the revised manuscript: page 4 line 56, “Ductal carcinoma in situ (DCIS) and invasive breast cancer were included in the study following NCCN criteria for further genetic evaluation.”

3. On Figure 2, “no PV or VUS” group should be renamed as Likely benign or no variants. Figure 2 might be improved by using color.

Author response: “no PV or VUS” group was renamed as “no variants”. Figure 2 was improved by using color.

4. In the Discussion expand discussion of the other Brazilian studies that have been published and how this study compares. Also include any additional studies of other nearby countries.

Author response: Most germline breast cancer studies in Latin America, including Brazil, focused on BRCA genes and used different testing approaches (founder mutation analysis, single gene analysis, copy number variation evaluation, multigene panel testing). For this reason, it was difficult to compare results from Brazil and nearby countries. The best scenario would be the comparison of our data to multigene panel testing studies. Nevertheless, after your suggestion we performed a new revision of the literature with the proposed view. Pubmed search pointed out only 3 studies with multigene panel testing (2 from Brazil and 1 from Colombia). (in the document "Response to reviewers" attached, there is a table with the information about these publications)

The reference 11 in our manuscript [Urbina-Jara LK, et al. Landscape of Germline Mutations in DNA Repair Genes for Breast Cancer in Latin America: Opportunities for PARP-Like Inhibitors and Immunotherapy. Genes (Basel). 2019 Oct 10;10(10):786], shows an excellent review of all the publications from Latin America countries related to BRCA and non-BRCA genes testing. Brazil is the country with most of the published data, in contrast with the majority of the other countries, many Latin America countries do not have any published data at all.

Here we illustrate the heterogeneity of genetic testing approaches in the most relevant Brazilian published studies:

Non-Multigene panel testing Brazilian BC data is illustrated in a table in the document attached "Response to reviewers".

We rewrote the discussion trying to incorporate suggestions.

Changes in the text of the revised manuscript: page 12 line 193-200, “Notwithstanding, there are global disparities in genetic testing accessibility.

Genetic testing is not accessible for the majority of the population from developing and underdeveloped countries. Although there is an international current debate about BC genetic testing based on clinical criteria versus BC universal testing (24–26), Brazil and other Latin America countries face limited access. The main reported barriers are related to the lack of structured genetic counseling and genetic testing networks, insufficient number of trained professionals in high risk cancer assessment, absence of genetic testing access in the public health system, limited health insurance coverage, costs of genetic testing and lack of national policies (27,28)”

Changes in the text of the revised manuscript: page 13-14 line 209-240, “The detection rate of actionable PVs varies widely depending on the selected studied population and the genetic testing approach (founder mutations, single gene analysis, copy number variation evaluation, multigene panel testing). The present cohort consisted of consecutive BC patients referred to genetic counseling selected due to the suspicion of hereditary BC. The overall detection rate of PVs was 20.5% (46/224). According to NCCN criteria, 85.3% (191/224) of our cohort met hereditary BC testing criteria, among these patients 23.6% (45/191) harbored PVs. Whereas, among 33 patients that did not met NCCN criteria, only one harbored a PV (3%). The detection rate of PVs was similar to other multigene panel testing studies based on hereditary breast and ovarian cancer (HBOC) criteria (5,12,13,30–32).

Fifty-seven percent of the patients with a positive test result harbored PVs in high-penetrance BC genes (41.3% in BRCA1/2, 17.4% in TP53, and 2.2% in PALB2), 15.2% in moderate penetrance BC genes (2.2% in ATM and 13% in CHEK2), and 23.9% in genes considered to be associated with a BC potential increased risk or an unknown risk. Nine percent of the patients (20/224) had actionable variants in non-BRCA genes, representing 43.5% of the positive results and 51.2% of the actionable variants.

The detection rate of PVs in moderate penetrance BC genes varies from 2 to 8% (5,32–34). Our cohort was enriched by PVs in moderate penetrance BC genes (15.2% of positive results, 9% of overall genetic tests). Another Brazilian study with individuals from the Northeast of the country also found a high prevalence of PVs (32% of positive results) in moderate penetrance BC genes (12). These findings suggest that Brazilian patients should have access to multigene panel testing.

Although there is a higher frequency of CHEK2 founder mutations (c.1100delC, c.470T>C) in European ancestry populations, these mutations were not observed in our cohort. Other Brazilian studies have also showed no enrichment of CHEK2 European founder mutations in BC Brazilian patients (35–38). The majority of CHEK2 PVs reported in our study affected RNA splicing (Table 2).

There are limited studies from Brazil and Latin American countries with BC germline characterization by multigene panel testing. A Brazilian BC study from the Northeast of the country showed a PV frequency of 17% (27/157), 68% (13/19) harbored a PV in BRCA1/2 genes and 32% (6/19) in moderate penetrance BC genes (12). Most of these patients were tested using a 33-gene panel. A research group from the Brazilian Southeast region performed a 21-gene panel in 95 women with a personal history of BC or HBOC clinical suspicion based on family history criteria (13). Twenty-three percent of the patients harbored a PV in BRCA1/2 and TP53 genes. Eighty-five women from Colombia, meeting HBOC criteria, had germline testing with a commercial 25-gene hereditary cancer panel (32). Twenty-two percent of the patients (19/85) harbored a PV in a cancer susceptibility gene.”

Changes in the text of the revised manuscript: page 15 line 275-283, ”This study had several limitations. It was retrospective and had a limited sample size compared to multigene testing studies around the world. However, it represents the largest Brazilian BC cohort from a single institution tested using a multigene panel for hereditary BC (12,13). It is also the first BC germline characterization from the Center-West of the country. These findings require validation in other cohorts. The study was conducted at a private cancer center and, for this reason, the assessed population may differ from that of the general community. Furthermore, it consisted of a high-risk population for hereditary cancer with a median age of 45 years at BC diagnosis, mostly composed of patients with premenopausal BC (68.7%), a positive family history for cancer (89.7%), and a personal history of multiple primary cancers (17.4%).”

5. Deposit sequence variants found into a publicly accessible database.

Author response: All variants were included in publicly accessible databases such as ClinVar from the National Center for Biotechnology information, by the commercial laboratories. Invitae also created a free access database called ClinVitae (clinvitae.invitae.com), which aggregates data from ClinVar (including variants contributed by Invitae), Emory Genetics Variant Classification catalog, ARUP Mutation Databases, Carvaer Mutation Database, Kathleen Cunninham Foundation Consortium for Research in Familial Breast Cancer. We also included in Table 2 and S4 the rsID related to the Single Nucleotide Polymorphism Database identification or the Clinvar variant identifier number (variation ID).

Section 2-Reviewer 1 comments to the authors:

Author response: First of all, we would like to thank you for all the relevant comments and suggestions that were essential for the manuscript improvement.

Author response: We rearranged the table´s information in alphabetic order according to the gene harboring the pathogenic variant described to facilitate comparison. The table was also included in the main text as Table 2.

b) In Figure 2. a straight category might be more adequate (like “Benign”) rather that “No PV or VUS”

Author response: “no PV or VUS” group was renamed as “no variants”.

c) There are representative studies in Brazil that were ignored in this MS, and the utility is obvious to contrasting the results of the cohorts. Although the authors disclosed:

It might be a good improvement to discuss comparing with larger cohorts, even of other regions, from the large country Brazil.

Author response: We agree with your comment. Nevertheless, most germline breast cancer studies in the Latin America, including Brazil, focused on BRCA genes and used different testing approaches (founder mutation, single gene analysis, copy number variation evaluation, multigene panel testing). We prepared a table, which was presented above (Section 1- Editor´s comments to the authors, comment number 4) to try to illustrate the most relevant Brazilian studies and multigene testing from Brazil and nearby countries.

As illustrated in the table, the Brazilian studies used different approaches of genetic testing. For this reason, it was difficult to compare results. Besides this, most Brazilian studies were conducted in the south and southeast of the country.

The best scenario would be the comparison of our data to other multigene panel testing studies. Pubmed search pointed out only 3 studies with multigene panel testing (2 from Brazil and 1 from Colombia). We rewrote the discussion trying to incorporate suggestions.

There was an intense racial interaction in Brazil, mainly Portuguese, African and Amerindian. There was also the influence of various flows of migrants coming to Brazil in the nineteenth and twentieth centuries, including French, Germans, Italians, Japanese, Middle Easterners (Turks and Arabs), Polish, Russians and Spanish. For this reason, each region has a different ancestry composition.

d) Discussing the application of the NCCN guidelines upon the findings in the present work, may be interesting

Author response: The indication of genetic testing according to the NCCN criteria was added to the results and to the discussion.

Changes in the text of the revised manuscript (Results): page 10 line 142-144, “A total of 191 patients (85.3%, 191/224) fulfilled NCCN hereditary BC testing criteria. Among these patients, 23.5% harbored PVs (45/191). In the group of patients that did not meet NCCN criteria, PV detection rate was 3% (1/33) (Appendix 3 in the Supplement).”

Changes in the text of the revised manuscript (Discussion): page 12 line 209-217, “The detection rate of actionable PVs varies widely depending on the selected studied population and the genetic testing approach (founder mutations, single gene analysis, copy number variation evaluation, multigene panel testing). The present cohort consisted of consecutive BC patients referred to genetic counseling selected due to the suspicion of hereditary BC. The overall detection rate of PVs was 20.5% (46/224). According to NCCN criteria, 85.3% (191/224) of our cohort met hereditary BC testing criteria, among these patients 23.6% (45/191) harbored PVs. Whereas, among 33 patients that did not met NCCN criteria, only one harbored a PV (3%). The detection rate of PVs was similar to other multigene panel testing studies based on hereditary breast and ovarian cancer (HBOC) criteria (5,12,13,30–32).”

e) A good idea is to deposit the variants in a public database for the free access to the worldwide scientific community

Author response: All germline genetic tests were performed by commercial molecular diagnostic laboratories, this information is detailed the S1 (supplement material). The majority of the genetic tests (196/219, 90%) were performed in the laboratory Invitae (San Francisco, California, USA). The variants are included in publicly accessible databases such as SNPdb and ClinVar by the commercial laboratories policies. We also included in the Table 2 and S4 the rsID related to the Single Nucleotide Polymorphism Database identification or the Clinvar variant identifier number (variation ID) of each detected pathogenic/likely pathogenic variant or variant of uncertain significance.

Section 3-Reviewer 2 comments to the authors:

Author response: First of all, we would like to thank you for all the relevant comments and suggestions that were essential for the manuscript improvement.

Reviewer #2: Methods: I would recommend using primary breast cancer as an inclusion criterion.

Author response: The National Comprehensive Cancer Network guidelines for Genetic/Familial High-Risk Assessment (https://www.nccn.org/professionals/physician_gls/default.aspx) include both invasive and ductal carcinoma in situ breast cancers as a criteria for further genetic evaluation (footnote d, version 1.2021, page 17 from NCCN guideline).

Results:

Study population: I would recommend excluding the remaining sixteen patients who had previously been diagnosed with other cancer because according to the title this paper is focused on breast cancer.

Author response: Multiple primary cancers is one of the most important clinical criteria for cancer predisposition syndromes screening. For this reason, the remaining sixteen patients that had been previously diagnosed with cancer, including thyroid cancer (6/16), melanoma (4/16), central nervous system (2/16), kidney (1/16), uterine cancer (1/16), lymphoma (1/16), and pancreatic cancer (1/16), were not excluded from the study population. Melanoma and pancreatic cancers are included in BRCA tumor spectrum. Uterine cancer (not specified as endometrial or cervix) as well as kidney cancer are included in Cowden syndrome tumor spectrum. Central nervous system, lymphoma and thyroid cancers are included in the Li-Fraumeni syndrome spectrum. Furthermore, four of these patients (25%, 4/16) harbored pathogenic variants. Multiple primary cancers was a criteria statistically significantly correlated with pathogenic variant detection.

Table 1: I would recommend including an extra column with the information of the total cohort for each item.

Author response: Total cohort is expressed in the edge of each column. We added the missing data information of each item.

Author response: We added in the article a table with the PVs detected (Table 2), as recommended. We added as Appendix 1 all the information about the performed genetic tests: commercial laboratories, number of genes in each panel, minimum of genes included in all the panels. The majority of the genetic tests (196/219, 90%) were performed in the commercial laboratory Invitae (San Francisco, California, USA), and included 80-84 genes.

Correlation between test positivity, germline genotype, and clinical data: I recommend including a table with the VUS detected in supplementary data.

Author response: We added a table with VUS detected in the supplementary material (S4) as recommended.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(61.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0247363.r003

Decision Letter 1

Amanda Ewart Toland

25 Jan 2021

PONE-D-20-32343R1

Germline molecular data in hereditary breast cancer in Brazil: lessons from a large single-center analysis.

PLOS ONE

Dear Dr. Sandoval,

1. Address the two minor points raised by reviewer 1.

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Reviewers' comments:

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Reviewer #1: All comments have been addressed

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Reviewer #1: Yes

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6. Review Comments to the Author

Reviewer #1: The MS “Germline molecular data in hereditary breast cancer in Brazil: lessons from a large single-center analysis” by Renata Lazari Sandoval, M.D., M.Sc et al, have made an amount of corrections.

1. One point that needs to be repaired is the sentence in page 14: “Even though most of the published data on hereditary BC among Latin American countries are from Brazil [11]” referring to the reference 11, should be rephrased since it might induce to a wrong concept that much of the contribution in Latin America is by the number of publications from Brazil, which is wrong. That review it is associated to PARP inhibitors and immunotherapy (stated in the title) which, besides, it is not reflecting the sense of this sentence. More important in that review, among most of the papers from Brazil, few ones refer to genes’ panels (a concept immediately expressed in the MS), many of the referenced Brazilian papers are biased by methodology limitations or patients´ selection, many refers to TP53 solely and, to be rescued, two references reflect the meaning of the sentence (ref 42 and ref 60). Importantly, published works in Latin America referred in the same review reflects the experience in a large series of cases that no one has reached at the year of publication (ref 98 and 100), these four publications are the ones that deserve a comment if that sentence is kept.

2. A reference should be added in the sentence pg. 26 line 263: “Our results are in concordance with previous reports stating that 13% of patients with multiple primary BC harbor PVs in BRCA2, CHEK2, BARD1, and RAD51C

Reviewer #2: (No Response)

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Reviewer #1: Yes: Angela SOLANO

Reviewer #2: Yes: Laura Cifuentes-C

PLoS One. 2021 Feb 19;16(2):e0247363. doi: 10.1371/journal.pone.0247363.r004

Author response to Decision Letter 1

4 Feb 2021

Dear Dr. Toland,

Thank you for giving us the opportunity to submit a second revised version of the manuscript “Germline molecular data in hereditary cancer in Brazil: lessons from a large single-center analysis”. We are grateful for the time and effort that you and the reviewers dedicated to providing feedback on our manuscript. All the comments provided the possibility of relevant improvements to our manuscript.

We have incorporated the suggestions made by the reviewer1. Authors´ responses are described in blue. Changes performed in the revised manuscript are highlighted in yellow. Page numbers refer to the revised manuscript file with tracked changes.

Reviewer #1:

1. One point that needs to be repaired is the sentence in page 3: “Even though most of the published data on hereditary BC among Latin American countries are from Brazil [11]” referring to the reference 11, should be rephrased since it might induce to a wrong concept that much of the contribution in Latin America is by the number of publications from Brazil, which is wrong. That review it is associated to PARP inhibitors and immunotherapy (stated in the title) which, besides, it is not reflecting the sense of this sentence. More important in that review, among most of the papers from Brazil, few ones refer to genes’ panels (a concept immediately expressed in the MS), many of the referenced Brazilian papers are biased by methodology limitations or patients´ selection, many refers to TP53 solely and, to be rescued, two references reflect the meaning of the sentence (ref 42 and ref 60). Importantly, published works in Latin America referred in the same review reflects the experience in a large series of cases that no one has reached at the year of publication (ref 98 and 100), these four publications are the ones that deserve a comment if that sentence is kept.

Author response: We fully agree and apologize for this misunderstanding. This comment was very important. We rephrased the sentence and included two references.

Changes in the text of the revised manuscript: page 3 line 40-41, “Even though there are published data on hereditary BC among Latin American countries [11,12], few studies have included non-BRCA genes and multigene panel testing [13,14]. Brazil is the largest country in South America and is the most genetically heterogeneous [14]. Most studies performed in Brazilian cohorts on hereditary breast cancer have been performed in the Southern and Southeastern parts of the country [12,15]. For this reason, epidemiological data about the prevalence of cancer predisposition syndromes in other parts of the country are needed.”

Author response: Thank you very much for this comment. We added this missing information and rephrased the data to better express our results in comparison to the literature. In our cohort 13/224 (5.8%) of patients had bilateral breast cancer (BC), seven patients did not harbor pathogenic variants (PVs). Six patients harbored PVs (6/13, 46%). Four out of thirteen (31%) patients harbored a PV in non-BRCA genes. Shin et al. (2020) described a cohort (n=496) with 15% of bilateral BC, 31,6% harbored PVs in non-BRCA genes (CHEK2, TP53, NBN, CDH1, MRE11A). Fanale et al. (2020) suggested that all bilateral BC patients should be offered multigene testing. They analysed a cohort of 139 cases of bilateral BC, 35.8% (19/53) harbored PVs in non-BRCA genes (PTEN, PALB2, CHEK2, ATM, RAD51C).

Changes in the text of the revised manuscript: page 15 line 262-266, “Patients with cancer predisposition syndromes have a higher risk of developing a second primary BC, especially for BRCA 1/2 mutation carriers [45]. Nevertheless, 8 - 36% of patients with bilateral BC harbor PVs in other genes beyond BRCA [32,47,48]. Bilateral BC represented 5.8% (13/224) of our cohort. Thirty-one percent (4/13) of patients with bilateral BC harbored PVs in non-BRCA genes (CHEK2, BARD1 and RAD51C). These results are in concordance with previous studies [32,47].”

Attachment

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PLoS One. doi: 10.1371/journal.pone.0247363.r005

Decision Letter 2

Amanda Ewart Toland

8 Feb 2021

Germline molecular data in hereditary breast cancer in Brazil: lessons from a large single-center analysis.

PONE-D-20-32343R2

Dear Dr. Sandoval,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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PLoS One. doi: 10.1371/journal.pone.0247363.r006

Acceptance letter

Amanda Ewart Toland

10 Feb 2021

PONE-D-20-32343R2

Germline molecular data in hereditary breast cancer in Brazil: lessons from a large single-center analysis.

Dear Dr. Sandoval:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Characteristics of genetic tests performed.

(DOCX)

Click here for additional data file.^{(13.5KB, docx)}

S2 Table. Clinical characteristics of patients with pathogenic and likely pathogenic variants.

(DOCX)

Click here for additional data file.^{(23.9KB, docx)}

S3 Table. Number of patients who meet current genetic testing criteria according to the genetic test result.

(DOCX)

Click here for additional data file.^{(12.9KB, docx)}

S4 Table. Variants of uncertain significance detected.

(DOCX)

Click here for additional data file.^{(23.7KB, docx)}

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(61.7KB, docx)}

Attachment

Submitted filename: Rebuttal letter-2nd revision.docx

Click here for additional data file.^{(27.5KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files. The original data is available in an excel file.

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PERMALINK

Germline molecular data in hereditary breast cancer in Brazil: Lessons from a large single-center analysis

Renata Lazari Sandoval

Ana Carolina Rathsam Leite

Daniel Meirelles Barbalho

Daniele Xavier Assad

Romualdo Barroso

Natalia Polidorio

Carlos Henrique dos Anjos

Andréa Discaciati de Miranda

Ana Carolina Salles de Mendonça Ferreira

Gustavo dos Santos Fernandes

Maria Isabel Achatz

Roles

Abstract

Introduction

Materials and methods

Fig 1. Cohort selection.

Statistical analyses

Results

Study population

Table 1. Baseline and demographic characteristics.

Frequency and spectrum of pathogenic germline variants

Fig 2. Genetic test results.

Table 2. Pathogenic and likely pathogenic variants detected.

Correlation between test positivity, germline genotype, and clinical data

Discussion

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Amanda Ewart Toland

Roles

Author response to Decision Letter 0

Decision Letter 1

Amanda Ewart Toland

Roles

Author response to Decision Letter 1

Decision Letter 2

Amanda Ewart Toland

Roles

Acceptance letter

Amanda Ewart Toland

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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