Germline Variant Spectrum in Southern Italian High-Risk Hereditary Breast Cancer Patients: Insights from Multi-Gene Panel Testing

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors present a study on breast cancer patients who underwent germline genetic testing in Southern Italy.  It is a well written study and adds to the body of literature in breast cancer patients.  A few comments to improve the manuscript.

1. The authors should justify the genes selected in the panel.  For example, CDH1 and PTEN are breast cancer gene, and not included.  Lynch syndrome is more associated with ovarian cancer rather than breast cancer, and EPCAM is not included. 

2.  The authors state that they use NCCN guidelines, but criteria do not seem to be quite the same as NCCN and they should state which year and version of NCCN guidelines they are using.  Also for these criteria, the authors should use more gender inclusive terms rather than Man and woman.

3. Not all the p. in the variant table are listed. 

4. The authors should comment on the recurrent BRCA1 founder variant.  It does not appear to be found frequently in their 254 patients, so why is it considered a founder variant?

5. did the bioinformatic analysis incorporate a copy number variant pipeline?

6. VUS table can be moved to supplement.  Similarly, are any of the VUS concerning or trending pathogenic?  Some discussion on that might be useful.

Author Response

Reviewer 1:

 

The authors present a study on breast cancer patients who underwent germline genetic testing in Southern Italy.  It is a well written study and adds to the body of literature in breast cancer patients.  A few comments to improve the manuscript.

 

 

1. The authors should justify the genes selected in the panel.  For example, CDH1 and PTEN are breast cancer gene, and not included.  Lynch syndrome is more associated with ovarian cancer rather than breast cancer, and EPCAM is not included. 

Reply: Thank you for your valuable comment regarding our gene panel selection.

We appreciate the opportunity to clarify our rationale. We would like to emphasize that our gene panel was designed to serve a broader diagnostic purpose beyond breast cancer testing alone, as our genetic testing service caters to patients with various tumor types.

This comprehensive approach reflects the complex nature of hereditary cancer syndromes and allows for more efficient diagnostic workflows in our clinical setting.

We acknowledge that established breast cancer predisposition genes such as CDH1 and PTEN were not included in this initial panel. It's important to note that we have since expanded our panel to include these and other relevant genes; however, these more recent data are not included in the present study to maintain temporal and methodological consistency in our analysis.

Regarding your specific query about Lynch syndrome genes, our findings align with the current understanding of their role in breast cancer risk. In our cohort, we did not detect any pathogenic variants in MLH1, MSH2, MSH6, or PMS2, which is consistent with the generally accepted view that Lynch syndrome genes play a limited role in breast cancer predisposition. However, we believe the inclusion of these genes remains valuable, particularly given the ongoing debate surrounding MSH6 potential role in breast cancer risk. As noted by the ClinGen Breast Cancer Gene Curation Expert Panel (https://search.clinicalgenome.org/kb/genes/HGNC:7329), MSH6 shows some evidence for breast cancer association, though its clinical significance continues to be a subject of discussion in the field.

 

 

2. The authors state that they use NCCN guidelines, but criteria do not seem to be quite the same as NCCN and they should state which year and version of NCCN guidelines they are using.  Also for these criteria, the authors should use more gender inclusive terms rather than Man and woman.

Reply:  We appreciate this thoughtful comment that allows us to clarify an important methodological aspect of our study. As NCCN guidelines have evolved significantly over the study period (2019-2023), we opted to consistently follow the AIOM (Italian Association of Medical Oncology) recommendations 2023, which specifically addresses the Italian healthcare system context and incorporates the most recent updates in genetic testing criteria. This choice ensures methodological consistency throughout the study period and better reflects our local healthcare setting.

We have modified the Methods section by removing the reference to NCCN and specifying our adherence to AIOM 2023 guidelines, which are specifically designed for and implemented within the Italian public healthcare system.

Importantly, we agree with the reviewer's suggestion regarding gender-inclusive language in the inclusion criteria section. We have revised this section by replacing "Women" and "Man" with "Individuals" throughout the selection criteria. These modifications maintain the clinical clarity necessary for genetic testing criteria while using more appropriate inclusive language.

 

3. Not all the p. in the variants table are listed. 

Reply: Thank you for this careful observation. Indeed, we have identified several protein descriptions (p.) missing in our variant tables. We have now added these protein-level annotations where they were inadvertently omitted. However, for intronic variants affecting splicing sites, the protein description is intentionally omitted as these variants are considered Loss-of-Function (LoF) variants where the exact protein product cannot be accurately predicted without targeted functional studies. This is particularly relevant for variants that may affect splicing efficiency or create alternative splice sites.

 

 

4. The authors should comment on the recurrent BRCA1 founder variant.  It does not appear to be found frequently in their 254 patients, so why is it considered a founder variant?

Reply: Thank you for your important question about the BRCA1 founder variant frequency in our cohort. The deletion c.4964_4982del in BRCA1, also known as 5083del19, was initially characterized as a founder mutation in South Italy (specifically in the Calabria region) by Baudi et al. [Baudi et al., 2001]. The classification as a founder mutation is based on several key observations:

The variant shows a distinct pattern of recurrence in the Calabrian geographic area and has been identified worldwide specifically in unrelated families of Calabrian origin. Importantly, molecular analyses have demonstrated that these carriers share a single haplotype, evidenced by the presence of a common allele in polymorphic microsatellite analysis. This molecular signature strongly suggests these families descended from a common ancient ancestor (identical by descent).

In our current study, we identified this deleterious germline mutation in 3/254 breast cancer cases, yielding a mutation rate of 1.18%. While this absolute frequency might appear low, it is important to note that these founder mutation carriers represent:

 

• 8% (3/34) of patients harboring pathogenic variants in the analyzed cancer susceptibility genes;

 

• 30% ( 3 of 10 patients ) of all BRCA1 mutation carriers in our cohort.

 

These proportions align with findings from other Italian studies [Figlioli et al., 2019], supporting the continued significance of this founder variant in our population.

A sentence has been added in the revised manuscript to clarify this point for the readers.

 

 

References

 

Baudi, F.; Quaresima, B.; Grandinetti, C.; Cuda, G.; Faniello, C.; Tassone, P.; Barbieri, V.; Bisegna, R.; Ricevuto, E.; Conforti, S.; et al. Evidence of a founder mutation of BRCA1 in a highly homogeneous population from southern Italy with breast/ovarian cancer. Hum. Mutat. 2001, 18, 163–164.

 

Figlioli G, De Nicolo A, Catucci I, Manoukian S, Peissel B, Azzollini J, Beltrami B, Bonanni B, Calvello M, Bondavalli D, Pasini B, Vignolo Lutati F, Ogliara P, Zuradelli M, Pensotti V, De Vecchi G, Volorio S, Verderio P, Pizzamiglio S, Matullo G, Aneli S, Birolo G, Zanardi F, Tondini C, Zambelli A, Livraghi L, Franchi M, Radice P, Peterlongo P. Analysis of Italian BRCA1/2 Pathogenic Variants Identifies a Private Spectrum in the Population from the Bergamo Province in Northern Italy. Cancers (Basel). 2021 Jan 30;13(3):532.

 

 

 

5. did the bioinformatic analysis incorporate a copy number variant pipeline?

Reply: Thank you for this important question regarding Copy Number Variant (CNV) analysis.

We acknowledge that our bioinformatic pipeline did not include CNV detection, which represents a limitation of our study. While we recognize that CNV analysis would provide a more comprehensive genetic screening, current literature suggests that large genomic rearrangements account for a smaller proportion of pathogenic variants compared to point mutations in breast cancer susceptibility genes. For instance, Large genomic rearrangements (LGRs) in BRCA1 are responsible for between 0 and 27% of all BRCA1 disease-causing mutations identified in numerous populations [Sluiter and van Rensburg, 2011].

We plan to implement CNVs analysis in future studies to ensure complete variant detection. This would enhance our understanding of the complete mutational spectrum in our population and potentially identify additional clinically relevant variants.

To clarify this point, the following sentence has been added in the Materials and Methods section of the revised manuscript: “The bioinformatic analysis pipeline used in this study did not include CNV detection algorithms ".

 

References

Sluiter, Michelle D., and Elizabeth J. van Rensburg. "Large genomic rearrangements of the BRCA1 and BRCA2 genes: review of the literature and report of a novel BRCA1 mutation." Breast cancer research and treatment 125 (2011): 325-349.

 

6. VUS table can be moved to supplement.  Similarly, are any of the VUS concerning or trending pathogenic?  Some discussion on that might be useful.

 

Reply: Thank you for this valuable suggestion regarding the VUS table placement and analysis. We agree with the reviewer and have relocated the VUS table to the supplementary materials as recommended.

Regarding the classification potential of our identified VUS, we conducted a thorough analysis of their characteristics and likelihood of pathogenicity. The majority of VUS in our cohort are missense variants, and most of them, particularly those in BRCA1/2, are located in coldspot regions, suggesting they are less likely to be reclassified as pathogenic [Dines et al., 2020].

However, our analysis revealed three variants that demonstrate characteristics suggesting potential pathogenicity. Two CHEK2 variants, c.911T>C (p.Met304Thr) and c.1160C>T (p.Thr387Ile), identified in two patients, are rare missense variants that have been found deleterious in a recent comprehensive functional study evaluating multiple CHEK2missense variants [Stolarova et al., 2023]. These variants are located in the catalytic region of CHEK2 and demonstrate deleterious effects across multiple in silico analyses.

Additionally, the ATM variant c.8560C>T (p.Arg2854Cys) warrants particular attention as it is a rare variant located in the kinase domain region. This variant shows deleterious scores across several bioinformatic predictors and has been found at significantly increased frequencies in prostate cancer patients compared with healthy controls [Paulo et al., 2018].

We have added a dedicated section in the Discussion of the revised manuscript addressing these variants and their potential clinical implications, providing a more comprehensive analysis of these potentially actionable VUS.

 

References

 

Dines JN, Shirts BH, Slavin TP, Walsh T, King MC, Fowler DM, Pritchard CC. Systematic misclassification of missense variants in BRCA1 and BRCA2 "coldspots". Genet Med. 2020 May;22(5):825-830. doi: 10.1038/s41436-019-0740-6.

 

Stolarova L, et al., ENIGMA CHEK2gether Project: A Comprehensive Study Identifies Functionally Impaired CHEK2 Germline Missense Variants Associated with Increased Breast Cancer Risk. Clin Cancer Res. 2023 Aug 15;29(16):3037-3050.

 

Paulo P, Maia S, Pinto C, Pinto P, Monteiro A, Peixoto A, Teixeira MR. Targeted next generation sequencing identifies functionally deleterious germline mutations in novel genes in early-onset/familial prostate cancer. PLoS Genet. 2018 Apr 16;14(4):e1007355. doi: 10.1371/journal.pgen.1007355.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigated the germline variants of 13 predisposition genes for breast cancer among 254 breast cancer patients from Southern Italy. The study design and findings were described well in the manuscript. However, there are some major concerns as below. BTW, It will be easier to review if line number can be added.

Abstract: Definition is needed for the “high-risk” in “…among high-risk breast cancer patients from Southern Italy.”

Introduction, 3^rd paragraph: Two large-scale population studies on high- and moderate-penetrance genes for breast cancer were not mentioned and summarized in the introduction. Below are the two studies along with a summarizing paper. In the manuscript, a better summarization of the current findings on high- and moderate-penetrance genes for breast cancer should be provided with citations of these three papers.

Hu C, Hart SN, Gnanaolivu R, et al. A Population-Based Study of Genes Previously Implicated in Breast Cancer. N Engl J Med. 2021;384(5):440-451. doi:10.1056/NEJMoa2005936

Breast Cancer Association Consortium, Dorling L, Carvalho S, et al. Breast Cancer Risk Genes - Association Analysis in More than 113,000 Women. N Engl J Med. 2021;384(5):428-439. doi:10.1056/NEJMoa1913948

Narod SA. Which Genes for Hereditary Breast Cancer? N Engl J Med. 2021;384(5):471-473. doi:10.1056/NEJMe2035083

Introduction: This study focused on germline variants profile among Southern Italian population, which was also emphasized in the title. However, there are not corresponding description about this population. What are the specific/unique characteristics of this population? Were they under-represented in previous genetic studies? Any previous studies on the germline variants among Southern Italian breast cancer patients? If so, were they limited with small sample size?

Methods, Patients Selection: The location where the patients were recruitment needs to be described.

Methods, Patients Selection: The format need to be revised. Number starts with “(1) Women with both breast and ovarian cancer”.

Results, Figure 1-2: The figure 1 and 2 are too vague. A higher resolution is needed for them.

Discussion: The novelty for Southern Italian population was not discussed. Findings of this study should be compared with previously reported germline variants spectrum among Northern Italian populations or general European descendants.

Author Response

Reviewer 2:

 

This study investigated the germline variants of 13 predisposition genes for breast cancer among 254 breast cancer patients from Southern Italy. The study design and findings were described well in the manuscript. However, there are some major concerns as below. BTW, It will be easier to review if line number can be added.

 

 

Abstract: Definition is needed for the “high-risk” in “…among high-risk breast cancer patients from Southern Italy.”

Reply: The authors thank the reviewer for this insightful comment that allows clarification of an important aspect of the study population. The term "high-risk" has been used to indicate "high genetic risk," as the study did not examine an unselected population but rather focused on individuals meeting specific personal and familial criteria that identify subjects with an increased probability of carrying germline predisposing mutations. The selection process involved comprehensive genetic counseling sessions where detailed personal and family cancer histories were collected and evaluated according to the Associazione Italiana di Oncologia Medica (AIOM). These criteria include: women with synchronous breast and ovarian cancer, breast cancer diagnosis ≤40 years, triple-negative breast cancer at any age, bilateral breast cancer ≤50 years, and male breast cancer patients.

This structured approach allowed the authors to restrict genetic testing to a population subset with the highest likelihood of harboring pathogenic germline variants. For greater precision and clarity, the authors have therefore modified the terminology from "high-risk" to "high genetic risk" in the abstract. The revised sentence in the abstract section now reads: "This study elucidates the prevalence and spectrum of germline variants in 13 cancer predisposition genes among high genetic risk breast cancer patients from Southern Italy." This modification provides better specificity while maintaining the concise nature appropriate for an abstract, and more accurately reflects the carefully selected nature of the study cohort.

 

 

 

 

 

 

Introduction, 3^rd paragraph: Two large-scale population studies on high- and moderate-penetrance genes for breast cancer were not mentioned and summarized in the introduction. Below are the two studies along with a summarizing paper. In the manuscript, a better summarization of the current findings on high- and moderate-penetrance genes for breast cancer should be provided with citations of these three papers.

 

Hu C, Hart SN, Gnanaolivu R, et al. A Population-Based Study of Genes Previously Implicated in Breast Cancer. N Engl J Med. 2021;384(5):440-451. doi:10.1056/NEJMoa2005936

 

Breast Cancer Association Consortium, Dorling L, Carvalho S, et al. Breast Cancer Risk Genes - Association Analysis in More than 113,000 Women. N Engl J Med. 2021;384(5):428-439. doi:10.1056/NEJMoa1913948

 

Narod SA. Which Genes for Hereditary Breast Cancer? N Engl J Med. 2021;384(5):471-473. doi:10.1056/NEJMe2035083

 

Reply: Thank you for the valuable feedback on our manuscript. We appreciate you highlighting the important studies on high- and moderate-penetrance genes for breast cancer that we had not previously included in the introduction. In the revised manuscript, we have updated the third paragraph of the introduction to provide a more comprehensive summary of the current research in this area. By incorporating these important references and summarizing the relevant research, we believe the introduction now provides a more comprehensive and up-to-date overview of the current state of knowledge in this field. We thank you for bringing these studies to our attention, as they have helped to improve the quality and completeness of our manuscript.

 

Introduction: This study focused on germline variants profile among Southern Italian population, which was also emphasized in the title. However, there are not corresponding description about this population. What are the specific/unique characteristics of this population? Were they under-represented in previous genetic studies? Any previous studies on the germline variants among Southern Italian breast cancer patients? If so, were they limited with small sample size?

Reply: The authors appreciate the reviewer's comment regarding the need to better characterize the Southern Italian population studied. The Italian population exhibits significant genetic heterogeneity due to both geographic and historical factors, with a distinct genetic gradient observed from North to South along the peninsula. The study population, specifically from Calabria in the "deep" South of Italy, presents unique genetic characteristics. This region is characterized by high rates of inbreeding and consanguinity, particularly in isolated mountain communities of the hinterland. These communities have historically maintained relatively homogeneous population sizes with low growth rates, creating distinct genetic pools. Historical migration patterns have also shaped this population's genetic structure. Over the past half-century, significant migration flows from this region toward Northern Italy and the Americas, driven by economic opportunities, have influenced the population dynamics.

Such demographic events can lead to founder effects, making this population particularly valuable for investigating genetic diseases [Fiorito et al., 2015].

The genetic distinctiveness of this population is evidenced by significant discoveries in hereditary breast cancer genetics. Notably, the first Italian founder mutations were identified in two geographically distinct populations: a BRCA2 founder mutation in Sardinia and a BRCA1 founder mutation in Calabria [Pisano et al., 2000; Baudi et al., 2001].

After our initial identification of the founder mutation, subsequent research has provided compelling evidence of its significance in the broader Southern Italian population. A study conducted in Sicily analyzing a consecutive series of Breast and Ovarian cancer patients validated the presence of this specific mutation, suggesting a founder effect extending beyond regional boundaries [Russo et al., 2007]. This finding was further substantiated by a comprehensive retrospective analysis encompassing a broader Sicilian cohort, which revealed that the c.4964_4982del BRCA1mutation occurred in 1.33% of all genetically tested probands, representing a substantial 17.6% of BRCA1-positive cases [Incorvaia et al., 2020].

This consistent detection pattern across different Southern Italian populations strongly supports the mutation's founder status and highlights its clinical significance in hereditary breast cancer risk assessment in this geographic region.

Two sentences have been added to the Introduction section of the revised manuscript to clarify the points requested by this reviewer.

 

References

 

Fiorito G, Di Gaetano C, Guarrera S, Rosa F, Feldman MW, Piazza A, Matullo G. The Italian genome reflects the history of Europe and the Mediterranean basin. Eur J Hum Genet. 2016 Jul;24(7):1056-62. doi: 10.1038/ejhg.2015.233. Epub 2015 Nov 11. PMID: 26554880; PMCID: PMC5070887.

 

Baudi, F.; Quaresima, B.; Grandinetti, C.; Cuda, G.; Faniello, C.; Tassone, P.; Barbieri, V.; Bisegna, R.; Ricevuto, E.; Conforti, S.; et al. Evidence of a founder mutation of BRCA1 in a highly homogeneous population from southern Italy with breast/ovarian cancer. Hum. Mutat. 2001, 18, 163–164.

 

Pisano, M.; Cossu, A.; Persico, I.; Palmieri, G.; Angius, A.; Casu, G.; Palomba, G.; Sarobba, M.G.; Rocca, P.C.; Dedola, M.F.; et al. Identification of a founder BRCA2 mutation in Sardinia. Br. J. Cancer 2000, 82, 553–559.

 

Russo, A.; Calò, V.; Agnese, V.; Bruno, L.; Corsale, S.; Augello, C.; Gargano, G.; Barbera, F.; Cascio, S.; Intrivici, C.; et al. BRCA1 genetic testing in 106 breast and ovarian cancer families from southern Italy (Sicily): A mutation analyses. Breast Cancer Res. Treat. 2007, 105, 267–276.

 

Incorvaia L, Fanale D, Badalamenti G, Bono M, Calò V, Cancelliere D, Castiglia M, Fiorino A, Pivetti A, Barraco N, Cutaia S, Russo A, Bazan V. Hereditary Breast and Ovarian Cancer in Families from Southern Italy (Sicily)-Prevalence and Geographic Distribution of Pathogenic Variants in BRCA1/2 Genes. Cancers (Basel). 2020 May 5;12(5):1158.

 

 

 

 

 

 

 

 

Methods, Patients Selection: The location where the patients were recruitment needs to be described.

Reply: We appreciate the reviewer's comment regarding the need to clarify our patient recruitment framework. Our study cohort was recruited through the Medical Genetics Unit at the Renato Dulbecco University Hospital in Catanzaro (Italy), which operates as a specialized genetic testing and counseling facility.

Patient referrals were coordinated through an established clinical network comprising:

 

1. Dulbecco Breast Cancer Unit;
2. Regional Oncology Units;
3. Primary care physicians.

 

The service area primarily encompasses the province of Catanzaro, with additional referrals from the neighboring provinces of Crotone, Cosenza, Vibo Valentia, and Reggio Calabria. This geographical distribution reflects the centralized nature of our genetic testing service within the Calabria region.

We have now integrated this detailed description into the Methods section (2.1. Patient selection) of the revised manuscript to provide a comprehensive overview of our recruitment strategy and geographical coverage. This information is crucial for understanding the representativeness of our cohort within the Southern Italian population.

 

Methods, Patients Selection: The format need to be revised. Number starts with “(1) Women with both breast and ovarian cancer”.

Reply: The authors thank the reviewer for identifying this formatting issue. This has been corrected in the revised manuscript, where the patient selection criteria are now properly numbered.

 

 

Results, Figure 1-2: The figure 1 and 2 are too vague. A higher resolution is needed for them.

Reply: The authors thank the reviewer for noting the image quality concerns. Both Figure 1 and Figure 2 have been revised in the updated manuscript with higher resolution to ensure optimal clarity and readability of all text elements.

 

 

Discussion: The novelty for Southern Italian population was not discussed. Findings of this study should be compared with previously reported germline variants spectrum among Northern Italian populations or general European descendants.

Reply: The European population exhibits considerable heterogeneity in its mutational spectrum across different countries (Janavičius, 2010). In Italy, previous studies have identified specific geographical regions where high-penetrance genes BRCA1 and BRCA2 contribute significantly to breast cancer susceptibility, with notable recurrence of specific variants attributable to founder effects, particularly in the Southern regions of Apulia, Sicily, and Calabria.

A comprehensive analysis of 2.026 hereditary breast and/or ovarian cancer (HBOC) patients from Apulia revealed that the BRCA1 c.5266dupC mutation accounts for 54.9% of 342 BRCA1 carriers (Patruno et al., 2021). Similarly, a retrospective study of 1.967 subjects from HBOC families in Sicily identified the BRCA1 c.4964_4982del19 variant as the most prevalent pathogenic variant in the Sicilian population, present in 18 families with 63 total carriers, representing 13% of all BRCA-positive carriers (Incorvaia et al., 2020).

In our Calabrian cohort, this same variant, previously described as a Calabrian founder mutation, accounts for 16.6% (3/18 patients) of identified BRCA1/2 mutations and 30% of all BRCA1 mutation carriers, despite our limited cohort size. It is noteworthy that our study focused exclusively on breast cancer patients, although this variant demonstrates high recurrence in ovarian cancer patients as well.

The advent of novel high-throughput technologies based on next-generation sequencing has enabled comprehensive testing of additional susceptibility genes. Notably, our study found that 54% of pathogenic variants occurred in genes other than BRCA1 and BRCA2. Further large-scale studies will be essential to establish the prevalence of these genetic variations and their territorial distribution. These findings may significantly influence mutation screening strategies, breast cancer prevention approaches, and personalized treatment protocols, particularly as genetic testing becomes increasingly integrated into routine oncological practice. Discussion has been now modified by adding this part in the revised manuscript.

 

References

 

Janavičius R. Founder BRCA1/2 mutations in the Europe: implications for hereditary breast-ovarian cancer prevention and control. EPMA J. 2010 Sep;1(3):397-412. doi: 10.1007/s13167-010-0037-y. Epub 2010 Jun 27.

 

Patruno M, De Summa S, Resta N, Caputo M, Costanzo S, Digennaro M, Pilato B, Bagnulo R, Pantaleo A, Simone C, Natalicchio MI, De Matteis E, Tarantino P, Tommasi S, Paradiso A. Spectrum of Germline Pathogenic Variants in BRCA1/2 Genes in the Apulian Southern Italy Population: Geographic Distribution and Evidence for Targeted Genetic Testing. Cancers (Basel). 2021 Sep 21;13(18):4714.

 

Incorvaia L, Fanale D, Badalamenti G, Bono M, Calò V, Cancelliere D, Castiglia M, Fiorino A, Pivetti A, Barraco N, Cutaia S, Russo A, Bazan V. Hereditary Breast and Ovarian Cancer in Families from Southern Italy (Sicily)-Prevalence and Geographic Distribution of Pathogenic Variants in BRCA1/2 Genes. Cancers (Basel). 2020 May 5;12(5):1158.

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

All my concerns have been addressed.