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作者 Stella S, Vitale SR, Martorana F, Massimino M, Pavone G, Lanzafame K, Bianca S, Barone C, Gorgone C, Fichera M, Manzella L
Stefania Stella, 1,2 Silvia Rita Vitale, 1,2 Federica Martorana, 1,2 Michele Massimino, 1,2 Giuliana Pavone, 3 Katia Lanzafame, 3 Sebastiano Bianca, 4 Chiara Barone, 5 Cristina Gorgone, 6 Marco Fichera, 6, 7 Livia Manzella1,21 Department of Clinical and Experimental Medicine, University of Catania, Catania, 95123, Italy;2 Center for Experimental Oncology and Hematology, AOU Policlinico “G.Rodolico – San Marco”, Catania , 95123, Italy; 3 Medical Oncology, AOU Policlinico “G. Rodolico – San Marco”, Catania, 95123, Italy; 4 Medical Genetics, ARNAS Garibaldi, Catania, 95123, Italy; 5 Medicine Genetics, ASP, Syracuse, 96100, Italy; 6 Department of Biomedical and Biotechnology Sciences, University of Catania, Medical Genetics, Catania, Italy, 95123; 7Oasi Research Institute-IRCCS, Troina, 94018, Italy Communications: Stefania Stella, tel +39 095 378 1946, email [email protected]; [email protected] Purpose: Germline mutations in BRCA1 and BRCA2 and established breast cancer (BC), ovary (OC) and other associated with a lifetime risk of cancer.Testing for the BRCA gene is key to assessing individual risk, as well as for finding prevention methods in healthy carriers and tailoring treatments in cancer patients.The prevalence of BRCA1 and BRCA2 alterations varies widely across geographic regions, and although data exist on BRCA pathogenic variants in Sicilian families, studies specifically targeting populations in eastern Sicily are lacking.The aim of our study was to investigate the incidence and distribution of BRCA pathogenic germline alterations in a cohort of BC patients from eastern Sicily and to assess their association with specific BC traits using next-generation sequencing.The presence of alterations correlated with tumor grade and proliferation index.RESULTS: Overall, 35 patients (9%) had a BRCA pathogenic variant, 17 (49%) in BRCA1 and 18 (51%) in BRCA2.BRCA1 alterations are prevalent in triple-negative BC patients, whereas BRCA2 mutations are more common in luminal BC patients.Compared with non-carriers, subjects with BRCA1 variants had significantly higher tumor grade and proliferative index.Conclusions: Our findings provide an overview of BRCA mutational status in BC patients from eastern Sicily and confirm the role of NGS analysis in identifying patients with hereditary BC.Overall, these data are consistent with previous evidence supporting BRCA screening for proper prevention and treatment of cancer in mutation carriers.
Breast cancer (BC) is the most common malignancy worldwide and the deadliest cancer in women.1 The biological features that determine BC prognosis and clinical behavior have been extensively studied and partially elucidated over time.In fact, several surrogate markers are currently used to classify BC into different molecular subtypes.They are estrogen (ER) and/or progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2) amplification, proliferation index Ki-67 and tumor grade (G).2 The combination of these variables identified the following BC categories: 1) Luminal tumors, showing ER and/or PgR expression, accounted for 75% of BCs.These tumors were further divided into Luminal A, when Ki-67 was below 20% and HER2 negative, and Luminal B, when Ki-67 was equal to or above 20% and in the presence of HER2 amplification, regardless of proliferation index; 2) HER2+ tumors that are ER and PgR negative but show HER2 amplification.This group accounts for 10% of all breast tumors; 3) Triple-negative breast cancer (TNBC), which does not show ER and PgR expression and HER2 amplification, accounts for about 15% of breast cancers.2-4
Among these BC subtypes, tumor grade and proliferation index represent cross-sectional biomarkers that are directly and independently associated with tumor aggressiveness and prognosis.5,6
In addition to the aforementioned biological features, the role of inherited genetic alterations leading to the development of BC has become increasingly important over the past few years.7 About 1 in 10 breast tumors are inherited due to germline alterations in specific genes.8 Two large epidemiological studies involving more than 180,000 women have recently identified a group of eight genes (ie, ATM, BARD1, BRCA1, BRCA2, CHK2, PALB2, RAD51C, and RAD51D) primarily responsible for hereditary BC.Among these genes, BRCA1 and BRCA2 (hereinafter referred to as BRCA1/2) showed the strongest correlation with the development of breast tumors.9-12 In fact, germline BRCA1/2 mutations significantly increase the lifetime risk of BC as well as other malignancies, including ovarian, prostate, pancreatic, colorectal, and melanoma.From age 13 to 80 years, the cumulative incidence of BC is 72% in women with a BRCA1 pathogenic variant (PV) and 69% in women with a BRCA2 PV.14
Notably, a recent publication suggests that BC risk depends on the type of PV.In fact, compared with pathogenic truncating variants, glaring missense variants, especially in the BRCA1 gene, are associated with a reduced risk of BC, especially in older women.15
The presence of BRCA1 or BRCA2 PV was associated with different biological and clinicopathological features.16,17 BRCA1-associated BCs tend to be clinically aggressive, poorly differentiated, and highly proliferative.These tumors are usually triple negative and have an early age of onset.Tumors that occur in BRCA2-mutated patients typically exhibit moderate to well-differentiated grades and variable proliferative indices.These tumors are more common in lumen B and usually occur in older adults.16-18 Notably, mutations in BRCA1 and BRCA2 increase sensitivity to specific treatments, including platinum salts and targeted drugs such as poly(ADP-ribose) polymerase inhibitors (PARPi).19,20
Over the past few years, the implementation of next-generation sequencing (NGS) in clinical practice has enabled an increasing number of BC patients to undergo molecular testing for cancer susceptibility syndromes, including BRCA1/2.21 Concurrently, definitions based on precise criteria regarding family history , demographic, and clinicopathological characteristics to better identify individuals worthy of BRCA1/2 testing.22,23 In this context, evidence is accumulating on BRCA1/2 screening in specific populations, highlighting differences across geographic regions.24–27 Although there are reports on the BC cohort in western Sicily, fewer data are available on BRCA1/2 screening in the eastern Sicily population.28,29
We describe here the results of germline BRCA1/2 screening in BC patients from eastern Sicily, further correlating the presence of BRCA1 or BRCA2 mutations with the main clinicopathological features of these tumors.
A retrospective study was carried out at the “Center for Experimental Oncology and Hematology” at the Policlinico Hospital.Rodolico – San Marco in Catania.From January 2017 to March 2021, a total of 455 patients with breast and ovarian, melanoma, pancreatic or prostate cancer were referred to our molecular diagnostic laboratory for BRCA/2 genetic testing.This study was conducted in accordance with the Declaration of Helsinki, and all participants provided written informed consent prior to molecular analysis.
Histological and biological characteristics (ER, PgR, HER2 status, Ki-67, and grade) of BC were assessed on core biopsy or surgical samples, considering only aggressive tumor components.Based on these characteristics, BCs were classified as follows: luminal A (ER+ and/or PgR+, HER2-, Ki-67<20%), luminal B (ER+ and/or PgR+, HER2-, Ki-67≥20%), luminal B-HER2+ (ER and/or PgR+, HER2+), HER2+ (ER and PgR-, HER2+) or triple negative (ER and PgR-, HER2-).
Before assessing BRCA1 and BRCA2 mutation status, a multidisciplinary team including an oncologist, a geneticist, and a psychologist conducted a tumor genetics consultation for each patient to determine the presence of BRCA1 and/or BRCA1. or individuals with a high risk of PV in the BRCA2 gene.Patient selection was performed according to the Italian Society of Medical Oncology (AIOM) guidelines and local Sicilian recommendations.30,31 These criteria include: (i) family history of known pathogenic variants in susceptibility genes (eg, BRCA1, BRCA2, TP53, PTEN); (ii) males with BC; (iii) those with BC and OC; (iv) women with BC <36 years, TNBC <60 years, or bilateral BC <50 years; (v) personal medical history of BC <50 years and at least one first-degree relative: (a) BC < 50 years; (b) non-mucinous and non-borderline OC of any age; (c) bilateral BC; (d) male BC; (e) pancreatic cancer; (f) prostate cancer; (vi) two or more Personal history of BC > 50 years and family history of BC, OC, or pancreatic cancer for relatives who are first-degree relatives to each other (including relatives with whom she is first-degree relatives); (vii) Personal history of OC and at least one first-degree relative: (a) BC <50 years; (b) NOC; (c) bilateral BC; (d) male BC; (vii) female with high-grade serous OC.
A 20 mL peripheral blood sample was obtained from each patient and collected into EDTA tubes (BD Biosciences).Genomic DNA was isolated from 0.7 mL whole blood samples using the QIAsymphony DSP DNA Midi kit Isolation Kit (QIAGEN, Hilden, Italy) according to the manufacturer’s instructions and passed through a Qubit® 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) Perform quantification. Target enrichment and library preparation are performed by the Oncomine™ BRCA Research Assay Chef, ready to be loaded into the Ion AmpliSeq™ Chef Reagents DL8 Kit for automated library preparation according to the manufacturer’s instructions.The kit consists of two multiplex PCR primer pools that can be used to study all BRCA1 (NM_007300.3) and BRCA2 (NM_000059.3) genes.Briefly, 15 µL of each diluted sample DNA (10 ng) was added to barcoded plates for library preparation and all reagents and consumables were loaded on the Ion Chef™ instrument.Automated library preparation and barcoded sample library pooling were then performed on the Ion Chef™ instrument.The number of prepared libraries was then assessed by a Qubit® 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.Finally, libraries are combined in equimolar ratios in Ion Chef™ library sample tubes (barcoded tubes) and loaded onto the Ion Chef™ instrument.Sequencing was performed using an Ion Torrent S5 (Thermo Fisher Scientific) instrument (Thermo Fisher Scientific) using an Ion 510 Chip (Thermo Fisher Scientific).Data analysis was performed by Amplicon Suite (SmartSeq srl) and Ion Reporter Software.
All variant nomenclature followed the current guidelines of the Human Genome Variation Consortium, available online (HGVS, http://www.hgvs.org/mutnomen).The clinical significance of BRCA1/2 variants was defined using the classification of the International Consortium ENIGMA (Evidence-Based Network for Interpreting Germline Mutant Alleles, https://enigmaconsortium.org/) and consulting different databases such as ARUP, BRCAEXCHANGE , ClinVar, IARC_LOVD, and UMD.The classification includes five distinct risk categories: benign (category I), likely benign (category II), variant of uncertain significance (VUS, category III), likely pathogenic (category IV), and pathogenic (category V).VarSome also analyzed the effect of mutations on protein structure and function, an informative tool with access to 30 databases.32
To assign potential clinical significance to each VUS, the following computational protein prediction algorithms were used: MUTATION TASTER, 33 PROVEAN-SIFT (http://provean.jcvi.org/index.php), POLYPHEN-2 (http:// /genetics.bwh.harvard.edu/pph2/) and Align-GVGD (http://agvgd.hci.utah.edu/agvgd_input.php).Variants classified as class 1 and 2 were considered wild type.
Sanger sequencing confirmed the presence of each pathogenic variant.Briefly, a pair of specific primers was designed for each detected variant by using the BRCA1 and BRCA2 gene reference sequences (NG_005905.2, NM_007294.3 and NG_012772.3, NM_000059.3, respectively).Therefore, targeted PCR was performed followed by Sanger sequencing.
Patients who tested negative for the BRCA1/2 gene were tested by multiplex ligation-dependent probe amplification (MLPA) according to the manufacturer’s instructions to assess the presence of large genomic rearrangements (LGR).Briefly, DNA samples are denatured and up to 60 BRCA1 and BRCA2 gene-specific probes are used, each detecting a specific DNA sequence approximately 60 nucleotides in length.Probe amplification products, consisting of a unique set of PCR amplicons, were then analyzed by capillary electrophoresis and by Cofalyser.Net software in conjunction with the appropriate batch-specific Cofalyser tables (www.mrcholland.com).
Selected clinicopathological variables (histological grade and Ki-67% proliferation index) were associated with the presence of BRCA1/2 PV, calculated using the Prism software v. 8.4 using Fisher’s exact test assuming p-value <0.05 to be significant.
Between January 2017 and March 2021, 455 patients were screened for germline BRCA1/2 mutations.Mutation testing was performed at the Policlinico Hospital’s Center for Experimental Oncology and Hematology.According to the Sicilian guideline (http://www.gurs.regione.sicilia.it/Indicep1.htm, N. 02-Venerdì 10 Gennaio 2020), the Rodolico of Catania – San Marco” overall, 389 patients There were breast cancer, 37 ovarian cancer, 16 pancreatic cancer, 8 prostate cancer and 5 melanoma. The distribution of patients according to cancer type and analysis results is shown in Figure 1.
Figure 1 shows a flow chart showing an overview of the study.Patients with breast, melanoma, pancreatic, prostate, or ovarian tumors were tested for mutations in the BRCA1 and BRCA2 genes.
Abbreviations: PVs, pathogenic variant; VUS, variant of uncertain significance; WT, wild-type BRCA1/2 sequence.
We selectively focused our studies on breast cancer cohorts.The patients had a median age of 49 years (range 23-89) and were predominantly female (n=376, or 97%).
Of these subjects, 64 (17%) had BRCA1/2 mutations and were all female.Thirty-five (9%) had PV and 29 (7.5%) had VUS.Seventeen (48.6%) of the 35 pathogenic variants occurred in BRCA1 and 18 (51.4%) in BRCA2, while 5 VUS occurred in BRCA1 (17.2%) and 24 (82.8%) in BRCA2 (Figures 1 and 2).LGR was not present in the MLPA analysis.
Figure 2. Analysis of BRCA1 and BRCA2 mutations in 389 breast cancer patients.(A) Distribution of pathogenic variants (PV) (red), variants of uncertain significance (VUS) (orange), and WT (blue) in 389 breast cancer patients; (B) 389 breast cancer patients Thirty-five (9%) had BRCA1/2 pathogenic variants (PVs).Among them, 17 (48.6%) were BRCA1 PV carriers (dark red) and 18 (51.4%) were BRCA2 carriers (light red); (C) 29 (7.5%) of 389 subjects carried VUS, 5 (17.2%) BRCA1 genes (dark orange) and 24 (82.8%) BRCA2 genes (light orange).
Abbreviations: PVs, pathogenic variant; VUS, variant of uncertain significance; WT, wild-type BRCA1/2 sequence.
We next investigated the prevalence of BC molecular subtypes in patients with BRCA1/2 PV.The distribution included 2 (5.7%) luminal A, 15 (42.9%) luminal B, 3 (8.6%) luminal B-HER2+, 2 (5.7%) HER2+ and 13 (37.1%) TNBC patients.Among BRCA1-positive patients, 5 (29.4%) had luminal B BC, 2 (11.8%) had HER2+ disease, and 10 (58.8%) had TNBC.Tumors without BRCA1 mutations were either luminal A or luminal B-HER2+ (Figure 3).In the BRCA2-positive subgroup, 10 (55.6%) tumors were luminal B, 3 (16.7%) were luminal B-HER2+, 3 (16.7%) TNBC and 2 (11.1%) were luminal A (Figure 3 ).No HER2+ tumors were present in this group.Thus, BRCA1 mutations are prevalent in TNBC patients, whereas BRCA2 alterations are predominant in lumen B individuals.
Figure 3 Prevalence of breast cancer subtypes in patients with pathogenic variants in BRCA1 and BRCA2.Histograms showing the distribution of BRCA1- (dark red) and BRCA2- (light red) PVs among molecular subtypes of breast cancer patients.Numbers reported within each box represent the percentage of patients with BRCA1 and BRCA2 PV for each breast cancer subtype.
Abbreviations: PVs, pathogenic variant; HER2+, human epidermal growth factor receptor 2 positive; TNBC, triple-negative breast cancer.
Subsequently, we assessed the type and gene localization of BRCA1 and BRCA2 PVs.In BRCA1 PV, we observed 7 single nucleotide variants (SNVs), 6 deletions, 3 duplications and 1 insertion.Only one mutation (c.5522delG) represents a new discovery.The most common BRCA1 PV detected in both subjects was c.5035_5039delCTAAT.This alteration involves a deletion of five nucleotides (CTAAT) in BRCA1 exon 15, resulting in the substitution of the amino acid leucine by tyrosine at codon 1679, and due to a translation frameshift with a predicted alternative stop codon lead to premature protein truncation.All other changes are detected in only one case.Notably, one of the reported PVs was located in the splice site consensus region (c.4357+1G>T) (Table 1).
Regarding BRCA2 PV, we observed 6 deletions, 6 SNVs and 2 duplications.None of the changes found are novel.Three mutations recurred in our population, c.428dup and c.8487+1G>A observed in 3 subjects, followed by c.5851_5854delAGTT retrieved in two cases.The c.428dup alteration involves a repeat of C in exon 5 of BRCA2, predicted to encode a truncated, non-functional protein.The c.8487+1G>A mutation occurs in the intronic region of BRCA2 intron 19 (± 1,2) and affects the splicing consensus sequence, resulting in altered splicing resulting in abnormal or absent protein.The c.5851_5854delAGTT pathogenic variant is due to a 4-nucleotide deletion from nucleotide positions 5851 to 5854 in the coding exon 10 of the BRCA2 gene and results in a translational frameshift with a predicted alternative stop codon (p.S1951WfsTer).Notably, as previously reported, both alterations c.631G>A and c.7008-2A>T were detected in the same patient.34 The first mutation involves the replacement of adenosine (A) in BRCA2 exon 7 with a guanine (G) containing nucleotide resulting in a change of valine to isoleucine at codon 211, isoleucine Amino acid is an amino acid with highly similar properties.This change affects normal mRNA splicing.The second variant is located in an intronic region and results in a double A to thymine (T) substitution before exon 13 of the gene encoding BRCA2.The c.7008-2A>T change may generate multiple transcripts of different lengths.Furthermore, in the group of BRCA2 PVs, 4 out of 18 changes (22.2%) were intronic.
We then mapped BRCA1/2 deleterious mutations in functional domains and protein-binding regions (Fig. 4).In the BRCA1 gene, 50% of PVs were located in the breast cancer cluster region (BCCR), while 22% of the mutations were located in the ovarian cancer cluster region (OCCR) (Fig. 4A).In BRCA2 PV, 35.7% of the variants were located in the BCCR region and 42.8% of the mutations were located in the OCCR (Fig. 4B).Next, we assessed the location of PV within the BRCA1 and BRCA2 protein domains.For the BRCA1 protein, we found three PVs in the loop and coiled coil domains, and two mutations in the BRCT domain (Fig. 4A).For the BRCA2 protein, 4 PVs mapped to the BRC repeat domain, while 3 intronic and 3 exonic changes were detected in the oligo/oligosaccharide-binding (OB) and tower (T) domains ( Figure 4B).
Figure 4 Schematic representation of BRCA1 and BRCA2 proteins and localization of pathogenic variants.This figure shows the distribution of BRCA1 (A) and BRCA2 (B) pathogenic variants in breast cancer patients.Exonic mutations are shown in blue, while intronic variants are shown in orange.The bar height represents the number of cases.The BRCA1 and BRCA2 proteins and their functional domains are reported.(A) The BRCA1 protein contains a loop domain (RING) and a nuclear localization sequence (NLS), a coiled-coil domain, an SQ/TQ cluster domain (SCD), and a BRCA1 C-terminal domain (BRCT).(B) The BRCA2 protein contains eight BRC repeats, a DNA-binding domain with a helical domain (Helical), three oligonucleotide/oligosaccharide-binding (OB) folds, a tower domain (T), and An NLS on the C side.Areas called the Breast Cancer Cluster Region (BCCR) and Ovarian Cancer Cluster Region (OCCR) are shown at the bottom.*Represents mutations that determine stop codons.
We then investigated BC clinicopathological features that might correlate with the presence of BRCA1/2 PV.Complete clinical records were available for 181 BRCA1/2-negative patients (non-carriers) and all carriers (n = 35).There was a correlation between tumor proliferation rate and grade.
We calculated the distribution of Ki-67 based on the median of our cohort (25%, range <10-90%).Subjects with Ki-67 < 25% were defined as “low Ki-67″, while individuals with values ​​≥ 25% were considered “high Ki-67″.Significant Ki-67 differences (p<0.01) were found between non-carriers and BRCA1 PV carriers (Fig. 5A).
Figure 5 Correlation of Ki-67 with grade distribution in breast cancer women with and without BRCA1 and BRCA2 PVs.(A) Boxplot showing median Ki-67 values ​​in 181 non-carrier BC patients versus BRCA1 (18) or BRCA2 (17) PV patients.P values ​​below 0.5 were considered statistically significant.(B) Histogram representing the assignment of BC cancer patients into histological grade groups (G2 and G3) according to BRCA1 and BRCA2 mutation status (WT subjects, BRCA1 and BRCA2 PVs carriers).
Likewise, we examined whether tumor grade correlated with the presence of BRCA1/2 PV.Since G1 BC was absent in our population, we divided the patients into two groups (G2 or G3).Consistent with the Ki-67 results, the analysis revealed a statistically significant correlation between tumor grade and BRCA1 mutation, with a higher proportion of G3 tumors in BRCA1 carriers compared to non-carriers (p<0.005) (Figure 5B ).
Advances in DNA sequencing technology have enabled unprecedented advances in BRCA1/2 genetic testing, with crucial implications for patients with a family history of cancer.To date, approximately 20.000 BRCA1/2 variants have been identified and classified according to the American Society of Medical Genetics 35 and the ENIGMA system.35,36 It is well known that the BRCA1/2 mutational spectrum varies widely across geographic regions.37 Within Italy, the rate of BRCA1/2 PVs ranged from 8% to 37%, showing wide intra-country variability.38,39 With a population of almost 5 million, Sicily is the fifth largest region in Italy in terms of the number of inhabitants.Although data exist on the distribution of BRCA1/2 in western Sicily, there is no extensive evidence in the eastern part of the island.
Our study is one of the first reports on the incidence of BRCA1/2 PV in BC patients in eastern Sicily.28 We focused our analysis on BC, as this is by far the most common disease in our cohort.
When testing 389 BC patients, 9% carried BRCA1/2 PVs, evenly distributed between BRCA1 and BRCA2.These results are consistent with those previously reported in the Italian population.28 Interestingly, 3% (13/389) of our cohort were male.This rate is higher than expected for male breast cancer (1% of all BCs),40 reflecting our selection of populations based on BRCA1/2 mutation risk.However, none of these men developed a BRCA1/2 PV, so they were candidates for further molecular analysis to rule out the presence of less common mutations such as PALB2, RAD51C and D, among others.Variants of uncertain significance were retrieved in 7% of subjects in which BRCA2 VUS was evident.Even this result is consistent with preexisting evidence.28,41,42
When we analyzed the distribution of BC molecular subtypes in BRCA1/2 mutant women, we confirmed known associations between TNBC and BRCA1 PV (58.8%) and between luminal B BC and BRCA2 PV (55.6%).16,43 The luminal A and HER2+ tumors in BRCA1 and BRCA2 PV carriers are consistent with existing literature data.16,43
We then focus on the type and location of the BRCA1/2 PV.In our cohort, the most common BRCA1 PV was c.5035_5039delCTAAT.Although Incorvaia et al. did not describe this variant in their Sicilian cohort, other authors have reported it as a germline BRCA1 PV.34 Several BRCA1 PVs were found in our cohort – eg c.181T>G, c.514del, c.3253dupA and c.5266dupC – which have been observed in Sicily.28 Of these, two BRCA1 founder mutations (c.181T>G and c.5266dupC) are commonly found in Ashkenazi Jews of Eastern and Central Europe (Poland, Czech), Slovenian, Austrian, Hungarian, Belarusian and German ), 44,45 and, in the United States and Argentina, was recently defined as a “recurrent germline variant” in Italian patients with BC and OC.The 34c.514del variant was previously identified in 8 breast cancer patients from northern Sicily in Palermo and Messina.Interestingly, even Incorvaia et al. found the c.3253dupA variant in some families in Catania.28 The most representative BRCA2 PVs are c.428dup, c.5851_5854delAGTT and the intronic variant c.8487+1G>A, which have been reported in more detail 28 in a patient in Palermo with c.428dup, c.5851_5854delAGTT PV was observed in households in northwestern Sicily, mainly in the Trapani and Palermo regions, whereas c.5851_5854delAGTT PV was observed in households in northwestern Sicily.The 8487+1G>A variant was more common in subjects from Messina, Palermo, and Caltanissetta.28 Rebbeck et al. previously described the c.5851_5854delAGTT alteration in Colombia.37 Another BRCA2 PV, c.631+1G>A, has been found in BC and OC patients from Sicily (Agrigento, Siracusa and Ragusa).28 Notably, we observed the coexistence of two BRCA2 variants (BRCA2 c.631G>A and c.7008-2A>T) in the same patient, which we assumed to be segregated in cis mode, as previously reported like that.34,46 These BRCA2 uble mutations are indeed frequently observed in the Italian region and have been found to introduce premature stop codons, affecting messenger RNA splicing and causing the BRCA2 protein to fail.47,48
We also mapped BRCA1 and BRCA2 PVs in putative OCCR and BCCR regions of protein domains and genes.These regions were described by Rebbeck et al. as risk areas for developing ovarian and breast cancer, respectively.49 However, the evidence regarding the association between the location of germline variants and breast or ovarian cancer risk remains controversial.28,50-52 In our population, BRCA1 PVs were predominantly located in the BCCR region, whereas BRCA2 PVs were predominantly located in the OCCR region.However, we were unable to find any association between putative OCCR and BCCR regions and BC features.This may be due to the limited number of patients with BRCA1/2 mutations.From a protein domain perspective, BRCA1 PVs are distributed along the entire protein, and BRCA2 alterations are preferentially found in the BRC repeat domain.
Finally, we correlated BC clinicopathological features with BRCA1/2 PV.Due to the limited number of patients included, we only found a significant correlation between Ki-67 and tumor grade.Although the assessment and interpretation of Ki-67 remains somewhat controversial, it is certain that high proliferative rates are associated with increased risk of disease recurrence and decreased survival.To date, the cutoff for distinguishing between “high” and “low” Ki-67 is 20%.However, this threshold does not apply to our BRCA1/2 mutation patient population, which has a median Ki-67 value of 25%.This trend in high Ki-67 rates can be explained by the prevalence in our luminal B and TNBC cohorts, of which few luminal A tumors were present.However, some evidence seems to suggest that a higher Ki-67 cutoff (25–30%) may better stratify patients according to their prognosis.53,54 From the results of our analysis, a significant correlation is not surprising.Occurs between high Ki-67 and grades and the presence of BRCA1 PV.In fact, BRCA1-related tumors are typical of TNBC and exhibit more aggressive features.16,17
In conclusion, this study provides a report on the mutational status of BRCA1/2 in a BC cohort from eastern Sicily.Overall, our findings are consistent with preexisting evidence, both in terms of mutation prevalence and clinicopathological features in BC.More studies in larger populations of BRCA1/2-mutant BC patients, such as using multigenome expanded mutational analysis, are warranted to assess the presence of PVs that are distinct and less frequent than BRCA1/2.This will allow the identification and proper management of the increasing number of subjects at increased risk of cancer due to genetic mutations.
We confirmed that patients signed informed consent to release their tumor samples anonymously for research purposes.All patients signed written informed consent according to the Declaration of Helsinki.According to the policy of AOU Policlinico “G.Rodolico – S.Marco”, this study was exempted from ethical review because the BRCA1/2 analysis was performed according to clinical practice and all patients gave written informed consent.Patients also consent to the use of their data for research purposes.
We thank Prof. Paolo Vigneri for his assistance in the care of breast cancer patients as requested by the Ethics Committee.
Federica Martorana reports honoraria from Istituto Gentili, Eli Lilly, Novartis, Pfizer.The other authors declare no conflicts of interest in this work.
1. Sung H, Ferlay J, Siegel RL, et al.Global Cancer Statistics 2020: GLOBOCAN estimates the incidence and mortality of 36 cancers in 185 countries around the world.CA Cancer J Clin.2021;71(3):209-249.doi: 10.3322/caac.21660