Two recurrent pathogenic/likely pathogenic variants in PALB2 account for almost half of PALB2 positive families in Slovenia

Two recurrent pathogenic/likely pathogenic variants in PALB2 account for almost half of PALB2 positive families in Slovenia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Two recurrent pathogenic/likely pathogenic variants in PALB2 account for almost half of PALB2 positive families in Slovenia Vita Andreja Mesarič, Ana Blatnik, Kristina Drusany Starič, Ksenija Strojnik, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4569442/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Dec, 2024 Read the published version in Human Genomics → Version 1 posted 11 You are reading this latest preprint version Abstract Introduction : The prevalence and spectrum of PALB2 pathogenic/likely pathogenic variants (PV/LPVs) may vary across different regions, and these have not yet been analysed and reported in Slovenian HBOC families. Methods : We performed a retrospective analysis of all 5099 consecutively tested individuals from 4610 families who fulfilled national criteria for HBOC-panel testing from January 2015 to January 2022. After genetic counselling, genetic testing with next generation sequencing was performed for all probands and cascade testing was offered to their blood relatives. Results : Among all probands tested 0.9% (40/4610) were PALB2 PV/LPV carriers. 14 different PALB2 PV/LPVs were detected, one of them was novel. Five PV/LPVs were found to be recurrent in Slovenian population with two most frequent being c.509_510del and c.1451T > A. Altogether, 61 individuals from 41 PALB2 positive families were identified, 43 being cancer patients. 27.9% PALB2 -positive cancer patients were diagnosed with more than one malignant tumour. We identified three double heterozygote carriers with additional PV/LPVs in ATM, CHEK2 and BRCA1. Discussion : This report provides the first comprehensive description of molecular and clinical characteristics of PALB2 carriers in Slovenia. The frequency of PALB2 pathogenic variants in the Slovenian HBOC accounts for 0.9% of all individuals tested for PVs in HBOC-related genes. Our study adds a novel recurrent mutation, which is unique to the Slovenian context and one PV/LPVs, which had not been reported in the literature so far. The results of our study add information on genotype and phenotype in PALB2- positive patients and may be used for population specific assessment. Ethics approval : The present study was approved by the National Ethics Committee and the Institutional Ethics Committee of the Institute of Oncology Ljubljana (0120–591/2020/3 on the 20th of January 2021). HBOC PALB2 breast cancer ovarian cancer risk-reducing salpingoophorectomy recurrent pathogenic variants Figures Figure 1 Figure 2 Background Cancer patients with suspected hereditary breast and ovarian cancer (HBOC) syndrome, along with their healthy relatives, can benefit from multigene panel testing. If they are identified as carriers of pathogenic/likely pathogenic variants (PV/LPVs) in high risk cancer genes, we may offer them prevention measures such as enhanced cancer surveillance and discuss risk reducing surgeries to lower cancer burden (Kapoor et al. 2015). Multigene panel testing may reveal not only PV/LPVs in BRCA1 and BRCA2 genes, but also in other HBOC -related genes. For over a decade, it has been recognized that germline PV/LPVs in PALB2 (partner and localizer of BRCA2) are associated with an increased risk of breast cancer (BC) (Rahman et al. 2007). Germline PV/LPVs in PALB2 are reported in up to 1% of BRCA1/2 negative breast cancer patients (Wu et al. 2020; Woodward et al. 2021). Other malignancies, such as male breast cancer (Pritzlaff et al. 2017), ovarian cancer (OC), pancreatic cancer (PaC) (Hu and Guo 2020), prostate cancer, colorectal, and gastric cancer (Foulkes 2008) were also reported in carriers of germline PV/LPVs in PALB2. The risk estimates are, however, based on analysis of small patients’ cohorts, the existing literature presents conflicting data, and the statistical evidence remains weak. Notably, the largest study so far by Yang et al. on 524 families from 21 countries, demonstrated a substantial association between germline PALB2 PV/LPVs and ovarian cancer (RR 2.91), pancreatic cancer (RR 2.37), and male breast cancer (RR 7.34) (Yang et al. 2020a). Nevertheless, further studies are necessary to enhance our understanding. The PALB2 gene encodes a protein that acts as a bridge between BRCA1 and BRCA2 proteins, playing a crucial role in homology-directed recombination DNA repair (Walsh 2015). There is some evidence that new targeted treatments, which are effective in BRCA1 and BRCA2 PV/LPV carriers (such as poly-ADP-ribose polymerase (PARP) inhibitors), are also effective in individuals with PALB2 PV/LPVs, which is unsurprising given the shared underlying biology (Castroviejo-Bermejo et al. 2018). Understanding an individual’s PALB2 mutation status is therefore essential for personalised management, not only in preventive setting, but also when making treatment decisions for these cancer patients. The prevalence and spectrum of PV/LPVs may vary across different regions due to ethnic differences. Quantifying cancer risks associated with specific PV/LPVs and understanding the biological characteristics of malignancies in carriers with these variants is important for establishment of targeted clinical guidelines. Institute of Oncology Ljubljana (IOL), where the study was conducted, is the principal national institution that supervises programs on comprehensive cancer care in Slovenia, which is a central European country with a population of two million. IOL offers cancer genetic counselling and genetic testing of high-risk individuals at the national level and therefore serves as a referral tertiary centre for the whole country. In the Slovene HBOC cohort the prevalence and spectrum of germline PALB2 PV/LPVs have not yet been analysed and reported. Our study aimed to describe these PV/LPVs in PALB2 and analyse the types of cancer and age of cancer diagnosis in PALB2 PV/LPV carriers. Methods Patients Multigene panel testing with next generation sequencing (NGS) for HBOC-related genes was introduced at the IOL in late 2014. The latest gene panel (in use since 2022) consists of nineteen genes: ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MLH1, MSH2, MSH6, NF1, PALB2, PMS2, PTEN, RAD51C, RAD51D, STK11, TP53 . A proband (an index case) was defined as a family member (usually an affected individual) through whom a family with a PV/LPV is ascertained. If a PV/LPV was diagnosed in at least two or more seemingly unrelated families, it was considered a recurrent variant. Our retrospective study group encompassed 5099 individuals (4564 females and 535 males) from 4610 HBOC families who underwent genetic counselling in our Cancer Genetics Clinic at the Institute of Oncology Ljubljana and were referred to germline genetic testing between January 2015 and January 2022. Family history data was collected from all tested families and all cancer diagnoses were verified in the Slovenian Cancer Registry. The registry contains data of all cancer diagnoses since 1950, when compulsory reporting of cancer diagnosis started in Slovenia. Positive family history for HBOC syndrome was defined as a family history of at least one first- or second-degree relative with breast, ovarian, prostate, or pancreatic cancer. We disregarded cases of non-melanoma skin cancer and cervical cancer in the analysis. Family members of carriers of PV/LPVs were offered either cascade genetic testing for known PV/LPV in the family or NGS panel testing in case they fulfilled the inclusion criteria for panel testing. If the tested individuals gave their consent to report secondary findings (reporting the finding of a PV/LPV not initially suspected), secondary findings were also reported according to the ACMG criteria (Richards et al. 2015). Patients who harboured variants of unknown significance were not included in the analysis. All participants provided written informed consent. The present study was approved by the National Ethics Committee and the Institutional Ethics Committee of the Institute of Oncology Ljubljana (0120–591/2020/3 on the 20th of January 2021). Research was conducted according to the 1975 Helsinki Declaration as revised in 1983 and the procedures used met the ethical standards of these bodies. DNA Extraction DNA was isolated from blood samples according to the established laboratory protocol as previously published (16). Next Generation Sequencing Next generation sequencing (NGS) was performed on Illumina MiSeqDx Sequencing System using TruSight Cancer Panel or TruSight Hereditary Panel (Illumina, San Diego, CA, USA) to enrich and sequence all translated exons and ± 25 bp flanking intronic regions of all HBOC panel genes. Bioinformatics and copy number analysis were performed as described by our group previously (Gornjec et al. 2019; Klančar et al. 2020). Germline variants were classified for their clinical importance according to ACMG/AMP guidelines (Plon et al. 2008; den Dunnen et al. 2016). Variants are described according to HGVS v20.05 nomenclature (den Dunnen et al. 2016). Our HBOC gene panel included 19 genes: ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MLH1, MSH2, MSH6, NF1, PALB2, PMS2, PTEN, RAD51C, RAD51D, STK11, TP53 . For PALB2 reference sequence LRG_308t1 (NM_024675.3) was used. All PV/LPV germline variants in HBOC genes detected by NGS were additionally confirmed by Multiplex Ligation-dependent Probe Amplification analysis or Sanger sequencing as described by our group previously (Stegel et al. 2022). Statistical Analysis Statistical analysis was performed using SPSS software (version 25). We used descriptive statistics to describe patients’ clinical, pathological, and genetic characteristics. Results Study cohort From January 1st 2015 to January 31st 2022, 5099 individuals (535 males and 4564 females) from 4610 families were tested for germline PV/LPVs in HBOC-related genes. The median age of individuals at the time of testing was 54 years. PV/LPV detection rate among 4610 tested probands/families In 19.1% (883/4610) of tested families a germline PV/LPV in HBOC-related genes was detected. BRCA1 PV/LPVs were detected in 8.4% (386/4610). Additionally, PV/LPVs were detected in BRCA2 in 4.9% (224/4610) of all probands, in CHEK2 in 1.8% (83/4610), in ATM in 1.5% (69/4610) and in PALB2 in 0.9% (40/4610). The frequency of all PV/LPVs in HBOC-related genes is presented in Fig. 1 . PALB2 PV/LPVs were detected in 1.0% of all BRCA1/2 negative families (40/4000). In 22 out of 883 (2.5%) families, probands were diagnosed with two PV/LPVs and in one family (0.1%) one proband was diagnosed with three PV/LPVs in the HBOC-related genes. PALB2 study cohort We identified PALB2 PV/LPVs carriers in 40 HBOC families. In the same period, one additional family was identified, where a PV/LPV was reported as a secondary finding when a male patient with gastric cancer was tested for Lynch syndrome. Our PALB2 positive cohort was therefore composed of 41 families. Within these 41 families, a total of 61 family members were identified as carriers of a PALB2 PV/LPV. Spectrum of PALB2 PV/LPVs in the Slovenian cohort of 41 PALB2 positive families We identified 14 different PALB2 PV/LPVs. PALB2 c.912 del p.(Val305*) had previously not been reported in the literature and was found in one proband (Table 1 ). Five PV/LPVs were recurrent. The two most frequent were c.509_510del and c.1451T > A, detected in 10 different families each, together encompassing almost half (20/41, 48.8%) of all PV/LPVs detected in our population. Table 1 Spectrum of PALB2 PV/LPVs in the Slovenian population. PALB2 PV/LPV type Previously reported ACMG/AMP and Variant class* Variant Type N of carriers N of families (% of all families) c.1451T > A p.(Leu484*) yes PV Class 5 nonsense 19 10 (24.5%) c.509_510del p.(Arg170Ilefs*14) yes PV Class 5 frameshift 14 10 (24.5%) c.1027C > T p.(Gln343*) yes PV Class 5 nonsense 8 6 (14.7%) c.172_175del p.(Gln60Argfs*7) yes PV Class 5 frameshift 5 4 (9.8%) c.3549C > G p.(Tyr1183*) yes LPV Class 4 nonsense 2 2 (4.9%) c.1317del p.(Phe440Leufs*12) yes PV Class 5 frameshift 3 1 (2.4%) c.1240C > T p.(Arg414*) yes PV Class 5 nonsense 3 1 (2.4%) c.48G > A p.(Lys16=) yes PV Class 5 synonymous, splicing 1 1 (2.4%) c.2192T > G p.(Leu731*) yes PV Class 5 nonsense 1 1 (2.4%) c.1676_1677delinsG p.(Gln559Argfs*2) yes PV Class 5 frameshift 1 1 (2.4%) c.3164dup p.(Tyr1055*) yes PV Class 5 nonsense 1 1 (2.4%) deletion of exons 11–12 c.(3113 + 1_3114-1)_(3350 + 1_3351-1)del p.? yes PV Class 5 multiple exon deletion 1 1 (2.4%) c.395del p.(Val132Alafs*45) yes PV Class 5 frameshift 1 1 (2.4%) c.912del p.(Val305*) no PV Class 5 nonsense 1 1 (2.4%) All PALB2 PV/LPVs 61 41 (100%) Legend: PV/LPV = pathogenic variant/likely pathogenic variant, SNV = single nucleotide variation, BC = breast cancer, OC = ovarian cancer, NET = neuroendocrine tumor, PaC = pancreatic cancer, f = female, m = male, ACMG/AMP classification (12) classification system proposed by Plon et al. (15). Cancer types diagnosed in PALB2 PV/LPV carriers Out of the 61 PALB2 -positive patients, 43 (70.5%) were diagnosed with at least one type of cancer (3 males, 40 females). The distribution of cancer types and ages at diagnosis are illustrated in Fig. 2 . The median age at the diagnosis of the first malignancy was 47 years (range 32 years – 69 years). Among 36 PALB2 positive BC (invasive and in situ) patients, 35 were females and one was male. The median age at BC diagnosis was 46.5 years, ranging from 32 years to 69 years. The median age at genetic testing was 51 years, ranging from 21 years – 79 years. Twelve different PV/LPVs were identified in PALB2 positive BC patients (Table 1 and Fig. 1 ). Male carriers with cancer Eight of the PALB2 carriers were male and three of them were diagnosed with one malignancy each: a carrier of PALB2 c.1027C > T p.(Gln343*) was diagnosed with BC at the age of 69, a carrier of PALB2 c.3549C > G p.(Tyr1183*) was diagnosed with pancreatic cancer at 45 years of age and a carrier of PALB2 c.2192T > G (p.Leu731*) was diagnosed with gastric cancer at 53 years of age. Clinical Characteristics of double heterozygotes Among PALB2 PV/LPV carriers, three patients were classified as double heterozygotes (DH). Their clinical characteristics are presented in Table 2 . Table 2 Characteristics of double heterozygotes. Patient (sex) PALB2 PV/LPV Other HBOC gene PV/LPV Cancer type (age at diagnosis) Histopathological Characteristics Patient 43 (f) deletion of exons 11–12 c.(3113 + 1_3114-1)_(3350 + 1_3351-1)del p.? ATM c.2413C > T p.(Arg805*) BC (48) poorly differentiated, bifocal, luminal B IDC Patient 17 (f) c.1451T > A p.(Leu484*) CHEK2 c.1100del p.(Thr367Metfs*15) BC (32) poorly differentiated, triple negative IDC Patient 42 (f) c.912del p.(Val305*) BRCA1 c.181T > G p.(Cys61Gly) BC (42) atypical medullary OC (57) HGSC BC (67) unknown histology PaC (69) adenocarcinoma Legend: PV/LPV = pathogenic variant/likely pathogenic variant, BC = breast cancer, OC = ovarian cancer, PaC = pancreatic cancer, HGSC = high-grade serous carcinoma, FIGO = the International Federation of Gynecology and Obstetrics, f = female, m = male. Multiple primary cancers In total, 12 out of 43 (27.9%) PALB2 positive patients with cancer were diagnosed with more than one malignant tumor. Among them, 7/12 had negative family history and 3/7 had their first cancer diagnosed after the age of 50 years. Median interval between first diagnosis and a new primary cancer was 8 years (range 1–18 years). Characteristics of patients with multiple primary malignancies are presented in Table 3 . Table 3 Characteristics of PALB2 positive patients with multiple primary malignancies in different organs. Patient (sex) Patient with PV/LPVs Type of Cancer (age at diagnosis) Family history (number of affected family members) Patient 33 (f) PALB2 c.1240C > T p.(Arg414*) melanoma (53), BC (56) positive (1) Patient 10 (f) PALB2 c.509_510del p.(Arg170Ilefs*14) papilla Vateri (56), fallopian tube (62) negative Patient 37 (f) PALB2 c.1676_1677delinsG p.(Gln559Argfs*2) melanoma (47), BC (48) positive (1) Patient 31 (f) PALB2 c.172_175del p.(Gln60Argfs*7) DCIS (49), NET origo ignota (67) negative Patient 42 (f) PALB2 c.912del p.(Val305*) BRCA1 c.181T > G p.(Cys61Gly) BC (42, 67), OC (57), PaC (69) positive (1) Patient 1 (f) PALB2 c.509_510del p.(Arg170Ilefs*14) BC (49, 53) positive (3) Patient 7 (f) PALB2 c.509_510del p.(Arg170Ilefs*14) BC (44, 52) negative Patient 5 (f) PALB2 c.509_510del p.(Arg170Ilefs*14) BC (58, 62) negative Patient 9 (f) PALB2 c.509_510del p.(Arg170Ilefs*14) BC (36, 48) negative Patient 18 (f) PALB2 c.1451T > A p.(Leu484*) BC (52, 60) negative Patient 36 (f) PALB2 c.1317del p.(Phe440Leufs*12) BC (51), DCIS (52) negative Patient 30 (f) PALB2 c.172_175del p.(Gln60Argfs*7) BC (40, 57) positive (2) Legend: PV/LPV = pathogenic variant/likely pathogenic variant, BC = breast cancer, OC = ovarian cancer, DCIS = ductal carcinoma in situ, NET = neuroendocrine tumor, PaC = pancreatic cancer; f - female As shown in Table 3 , one carrier of PALB2 PV/LPV, who also harboured a BRCA1 PV/LPV, was diagnosed with four primary cancers: twice with BC (at 42 and 67 years), a high-grade serous carcinoma of the ovary (age at diagnosis 57) and a pancreatic adenocarcinoma (age at diagnosis 69). Additionally, two PALB2 positive patients both developed BC (aged 48 and 56, respectively) and melanoma (aged 47 and 53, respectively). PALB2 positive carriers without cancer diagnosis Our cohort of PALB2 PV/LPV carriers without a cancer diagnosis consisted of 18 individuals from 11 families. Thirteen were female and five were male. Two of them were identified through panel testing and 16 by cascade testing. Median age at genetic testing was 44 years (range 21 – to 75 years). Among these individuals, PALB2 c.1451T > A p.(Leu484*) PV/LPV was found in 11 individuals from six families, PALB2 c.509_510del (p.(Arg170Ilefs14)) was found in two individuals from two different families, PALB2 c.1317del p.(Phe440Leufs12) was found in two individuals from the same family, and one individual was identified with each of the following PV/LPVs: PALB2 c.1240C > T p.(Arg414*), PALB2 c.1027C > T p.(Gln343*), and PALB2 c.172_175del p.(Gln60Argfs*7). Discussion Having epidemiological data on the frequency and spectrum of germline PV/LPVs associated with different hereditary cancers is of the utmost importance for every country aiming to organize an optimal cancer prevention programme and optimize cancer patients’ management. In the Slovene population, data on the occurrence of germline PV/LPVs in BRCA1 and BRCA2 genes and the clinicopathological characteristics of malignancies in those patients, have already been reported (20). Additionally, the spectrum of BRCA1 and BRCA2 PV/LPVs in male breast cancer patients, and characteristics of breast cancer patients with CHEK2 PV/LPVs have been studied (Besic et al. 2008; Krajc et al. 2014; Cvelbar et al. 2017; Nizic-Kos et al. 2021). PALB2 is one of the most common HBOC-related genes, found in approximately 1% of BRCA1/ 2 negative BC patients (Wu et al. 2020; Woodward et al. 2021) and is highly penetrant (Yang et al. 2020b). Nevertheless, very little is known about the characteristics of carriers of PALB2 PV/LPVs and the clinicopathological characteristics of tumors diagnosed in these patients. This gap in knowledge underscores the importance of further research into PALB2-associated cancers, not only for specialized institutions but also for primary care physicians. Detection rate and spectrum of PALB2 PV/LPVs Among all families tested with HBOC panel, we identified at least one germline PV/LPV in one of the genes in 19.1%. PALB2 was the fifth most commonly mutated HBOC-related gene (following BRCA1, BRCA2, CHEK2 and ATM ). PV/LPVs in PALB2 represent 0.9% of all individuals tested, which was expected as it had previously been reported that 0.2–0.9% of women with breast cancer who undergo genetic testing will carry germline PV/LPV in PALB2 (Hu et al. 2021). In our cohort the frequency of PV/LPVs in more than one gene from the HBOC panel in tested individuals is 2.6% (23/883) and is higher than the one reported in the literature, where we can find data ranging from 0.5 to 1% (Rosenthal et al. 2017; Shao et al. 2020). That may be due to the high prevalence of recurrent PV/LPVs in our population (Krajc et al. 2008; Stegel et al. 2011; Krajc et al. 2014). In 41 families 14 different PALB2 PV/LPVs were detected. PALB2 c.912 del p.(Val305*) had not been reported previously and was found in one proband. Newly described PALB2 PV/LPVs in patients with BC are important since they can contribute to international databases and patients may benefit from prevention and treatment options. Recurrent PV/LPVs in PALB2 in the Slovenian population Five PV/LPVs in PALB2 were recurrent (diagnosed in at least two or more seemingly unrelated families) in our population, with PALB2 c.509_510del and PALB2 c.1451T > A being the two most frequent, detected in 10 different families each, and together encompassing almost half (20/41 or 48.8%) of all detected PV/LPVs in our population. Recurrent PALB2 PV/LPVs have been reported in other populations as well, such as those from Argentina (Gonzalez et al. 2022), Finland (Erkko et al. 2007) and Poland (Noskowicz et al. 2014). PALB2 c.509_510del has been described by Noskowitz et al. as being present in about 1 in 400 unselected breast cancer patients from Central Europe (Germany) and Eastern Europe (Belarus, Russia) (Noskowicz et al. 2014). PALB2 c.509_510del has also been described as a recurrent mutation in BC and OC patients from Poland (Kluska et al. 2017). We found no reports on the presence of PALB2 c.509_510del in Western European, Asian or American populations. While the Slovenian language does belong to the South Slavic language group, genetic studies have revealed close genetic affiliations with West Slavic populations, such as Poles, suggesting a common Slavic ancestor originating from the Dnieper basin (Zupan et al. 2013). We found no genotype-phenotype correlation studies for this specific PV. Of note, three patients from our cohort (33.3%) were diagnosed with a metachronous contralateral BC, one additional (11.1%) with a metachronous contralateral and ipsilateral BC. Additionally, we found two cases of ovarian cancer (10.5% of PALB2 c.509_510delGA carriers) with a median age at diagnosis 56.5 years (range 51 years – 62 years), one case of carcinoma of the papilla Vateri and one malignant melanoma. Based on our data BC patients harbouring PALB2 c.509_510del are at high risk of developing a second BC and OC, making them high-risk group among PALB2 PV carriers. PALB2 c.1451T > A has not yet been described as a recurrent mutation in any population in the literature, however it has been found in nineteen individuals from ten different families in our cohort, which makes it a unique recurrent mutation in the Slovenian population. Among nineteen carriers of the above-mentioned PV, eight were diagnosed with breast cancer, with a median age at diagnosis 49 years. While it is challenging to establish genotype-phenotype correlation, this information could still be valuable in everyday clinical practice and may be included in the studies with bigger sample sizes. Malignancies among PALB2 PV/LPV carriers It has been known for more than a decade that PALB2 PV/LPVs increase BC risk (Hamdan and Nowak 2022). The risk is 2–30 times higher than in the general population, depending on the type of PV/LPV, age, and family history and PALB2 is considered a high-penetrance susceptibility gene for BC (Antoniou et al. 2014). As expected, the most common malignancy diagnosed in our cohort was BC in 36 patients, which confirms this association. Recent research has strengthened the correlation between PALB2 PV/LPVs and ovarian cancer. Yang et al. demonstrated a substantial association between germline PALB2 PV/LPVs and ovarian cancer with a risk ratio of around 3 and the lifetime risk of OC estimated to be around 3–5%(Yang et al. 2020a). In our cohort, four patients were diagnosed with a high-grade serous carcinoma of the ovary or the fallopian tube and one with a borderline ovarian carcinoma. There were no cases of primary peritoneal serous carcinoma. OC was the second most prevalent malignancy in our study. Two PALB2 PV/LPV carriers in our cohort (2/61, 3.3%) were diagnosed with PaC and additional carrier with carcinoma of Papilla Vateri. Indeed there is emerging evidence that PALB2 PV/LPVs predispose patients to pancreatic cancer. It is estimated that 3–4% of patients with famillial PaC are expected to harbour PALB2 PV/LPV. In the newest version of the National Comprehensive Cancer Network (NCCN) Guidelines enhanced screening not only for BC (with possible risk reducing surgeries), but also for OC (risk reducing surgery is offered as an option) and PaC is recommended (Clinical et al. 2024). Additionally, we have identified 3 cases of malignant melanoma, 1 NET and 1 gastric cancer in our cohort, there is, however, insufficient evidence to draw a conclusion about these types of malignancies. Double heterozygotes The increased use of multigene panels in the recent years has led to identification of individuals harbouring more than one PV/LPV in cancer susceptibility genes, although the data is still scarce. Double heterozygous PV in BRCA1/2 are identified in 0.3% of Ashkenazi and are very rare in other populations. Lavie et al. suggest that DH PV in BRCA1/2 in females of Ashkenazi Jewish heritage does not seem to cause a more severe phenotype than in cases where only one of the genes is implicated (Lavie et al. 2011). The results of the studies on DH VP in HBOC-related genes in other populations have been conflicting and inconclusive. To the best of our knowledge no research has been conducted on DH with one of the variants being PALB2 PV/LPV. A case report by Agiannitopoulos described a female patient, who was diagnosed at the age of 42 with endometrial cancer. DH in PALB2 and MSH explained the remarkable family history of ovarian, breast, kidney and colorectal cancer and consequently the surveillance of family members was adjusted (Agiannitopoulos et al. 2020). There is emerging evidence of multiplicative effect of presence of PV/LPVs in more than one cancer susceptibility gene. Heidemann et al. showed that Caucasian female DH for BRCA1/2 seem to develop BC at a younger age and have more severe disease than carriers of a single BRCA1/2 PV/LPV (Heidemann et al. 2012). Similarly, Sokolenko et al. pointed out that the presence of additional gene defect in female BRCA1 PV carriers may further increase their chances for cancer (Sokolenko et al. 2014). We have identified three DH carriers in our PALB2 cohort with additional PV/LPVs in ATM , CHEK2 , and BRCA1 . The clinical presentations varied. DH patients with PV/LPVs in PALB2 and ATM or CHEK2 were diagnosed with BC at 48 and 32 years, respectively. Patient who was found to harbour PALB2 and BRCA1 PV/LPV was diagnosed with 4 malignancies: two BC, OC and PaC, supporting the multiplying effect of DH. Genetic counselling for DH carriers is complex and further studies are required to elucidate its biological effect. Strengths and limitations of our study There are several limitations of our study. The expected population burden of PALB2 PV/LPVs in the Slovenian population is 0.13% (Kotnik et al. 2023). The absolute number of included PV/LPV carriers was small (61). 43 (70.5%) had a cancer diagnosis. However, our cohort represents 2% of the expected population of PALB2 PV/LPV carriers in Slovenia. Also, in comparison to the literature, where mostly case series are described, this is a large cohort of PALB2 PV/LPV carriers. The subgroups of patients with different PV/LPV were very small, therefore we were not able to analyse them separately. The retrospective collection of data is always unfavourable, since data can be missing or inappropriately understood, however as it can be seen from our data, the information regarding patients’ and tumours’ characteristics was complete for patients in our study. Its strength lies in a reliable family history, with information obtained from the Slovenian National Cancer Registry. It is one of the oldest Registries in Europe, where the diagnoses are cross-checked with histopathological reports. In cases of rare genetic diseases large multicentric studies are required to achieve a substantial number of patients and we hope these will be able to benefit from our cohort of PALB2 PV/LPV carriers. Conclusions This report provides the first comprehensive insight into the genotype and phenotype of PALB2 PV/LPV carriers in Slovenia. The frequency of PALB2 PV/LPV in Slovenia is consistent with rates reported in other countries, accounting for 0.9% of all individuals tested for PVs in HBOC-related genes. Notably, we identified two recurrent PALB2 PV/LPVs within our population, collectively encompassing nearly half of all affected families. Of particular interest is the PALB2 c.1451T > A variant, which has not been previously documented as a recurrent mutation in any population, rendering it unique to Slovenia The most common malignancy in our cohort was as expected BC, followed by OC and PaC, which adds to the existing evidence of PALB2 involvement in the pathogenesis of thesecancer types. Despite the rarity of PALB2 carriers, who also carry PV/LPVs in other HBOC-related genes, we have identified three such individuals, and studying their disease characteristic can help elucidate the biological effect of being a DH. Overall, the results of our study provide valuable genotype and phenotype data from PALB2 positive patients which may already be utilized in a population specific assessment. Declarations Funding The study was supported by the Slovenian Research Agency, program number P3-0289. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions Conceptualization: Vita Andreja Mesarič, Mateja Krajc, Methodology: Mateja Krajc, Kristina Drusany Starič, Simona Hotujec; Formal analysis and investigation: Vita Dragoš Šetrajčič, Petra Škerl, Srdjan Novaković, Vida Stegel; Writing - original draft preparation: Vita Andreja Mesarič; Writing - review and editing: Mateja Krajc, Ana Blatnik, Ksenija Strojnik, Kristina Drusany Starič. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics approval All participants provided written informed consent. The present study was approved by the National Ethics Committee and the Institutional Ethics Committee of the Institute of Oncology Ljubljana (0120-591/2020/3 on the 20 th of January 2021). Research was conducted according to the 1975 Helsinki Declaration as revised in 1983 and the procedures used met the ethical standards of these bodies. References Agiannitopoulos K, Papadopoulou E, Tsaousis GN, Pepe G, Kampouri S, Patsea E, Lypas G, Nasioulas G (2020) Report of a germline double heterozygote in MSH2 and PALB2. 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Cite Share Download PDF Status: Published Journal Publication published 18 Dec, 2024 Read the published version in Human Genomics → Version 1 posted Editorial decision: Revision requested 15 Jul, 2024 Reviews received at journal 14 Jul, 2024 Reviews received at journal 14 Jul, 2024 Reviews received at journal 04 Jul, 2024 Reviewers agreed at journal 13 Jun, 2024 Reviewers agreed at journal 13 Jun, 2024 Reviewers agreed at journal 13 Jun, 2024 Reviewers invited by journal 13 Jun, 2024 Editor assigned by journal 12 Jun, 2024 Submission checks completed at journal 12 Jun, 2024 First submitted to journal 12 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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(N=4610).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4569442/v1/791725b25de592065a8afac9.png"},{"id":59871781,"identity":"b4253f70-bb59-4e63-83b9-1361acfa6f80","added_by":"auto","created_at":"2024-07-08 17:09:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":122097,"visible":true,"origin":"","legend":"\u003cp\u003ePV/LPV variant, type of cancer and age at diagnosis in \u003cem\u003ePALB2 \u003c/em\u003ePV/LPV carriers.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4569442/v1/88f8a340a5030e6c7a9ed187.png"},{"id":72201990,"identity":"40efa24d-fcef-464a-97f8-3f3e948d2f58","added_by":"auto","created_at":"2024-12-23 16:13:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":948737,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4569442/v1/1ec0720c-3d1d-405a-9c7d-a1042e59b520.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Two recurrent pathogenic/likely pathogenic variants in PALB2 account for almost half of PALB2 positive families in Slovenia","fulltext":[{"header":"Background","content":"\u003cp\u003eCancer patients with suspected hereditary breast and ovarian cancer (HBOC) syndrome, along with their healthy relatives, can benefit from multigene panel testing. If they are identified as carriers of pathogenic/likely pathogenic variants (PV/LPVs) in high risk cancer genes, we may offer them prevention measures such as enhanced cancer surveillance and discuss risk reducing surgeries to lower cancer burden (Kapoor et al. 2015). Multigene panel testing may reveal not only PV/LPVs in \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e genes, but also in other HBOC -related genes.\u003c/p\u003e \u003cp\u003eFor over a decade, it has been recognized that germline PV/LPVs in \u003cem\u003ePALB2\u003c/em\u003e (partner and localizer of BRCA2) are associated with an increased risk of breast cancer (BC) (Rahman et al. 2007). Germline PV/LPVs in \u003cem\u003ePALB2\u003c/em\u003e are reported in up to 1% of \u003cem\u003eBRCA1/2\u003c/em\u003e negative breast cancer patients (Wu et al. 2020; Woodward et al. 2021). Other malignancies, such as male breast cancer (Pritzlaff et al. 2017), ovarian cancer (OC), pancreatic cancer (PaC) (Hu and Guo 2020), prostate cancer, colorectal, and gastric cancer (Foulkes 2008) were also reported in carriers of germline PV/LPVs in \u003cem\u003ePALB2.\u003c/em\u003e The risk estimates are, however, based on analysis of small patients\u0026rsquo; cohorts, the existing literature presents conflicting data, and the statistical evidence remains weak. Notably, the largest study so far by Yang et al. on 524 families from 21 countries, demonstrated a substantial association between germline \u003cem\u003ePALB2\u003c/em\u003e PV/LPVs and ovarian cancer (RR 2.91), pancreatic cancer (RR 2.37), and male breast cancer (RR 7.34) (Yang et al. 2020a). Nevertheless, further studies are necessary to enhance our understanding.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ePALB2\u003c/em\u003e gene encodes a protein that acts as a bridge between BRCA1 and BRCA2 proteins, playing a crucial role in homology-directed recombination DNA repair (Walsh 2015). There is some evidence that new targeted treatments, which are effective in \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e PV/LPV carriers (such as poly-ADP-ribose polymerase (PARP) inhibitors), are also effective in individuals with \u003cem\u003ePALB2\u003c/em\u003e PV/LPVs, which is unsurprising given the shared underlying biology (Castroviejo-Bermejo et al. 2018). Understanding an individual\u0026rsquo;s \u003cem\u003ePALB2\u003c/em\u003e mutation status is therefore essential for personalised management, not only in preventive setting, but also when making treatment decisions for these cancer patients.\u003c/p\u003e \u003cp\u003eThe prevalence and spectrum of PV/LPVs may vary across different regions due to ethnic differences. Quantifying cancer risks associated with specific PV/LPVs and understanding the biological characteristics of malignancies in carriers with these variants is important for establishment of targeted clinical guidelines.\u003c/p\u003e \u003cp\u003e Institute of Oncology Ljubljana (IOL), where the study was conducted, is the principal national institution that supervises programs on comprehensive cancer care in Slovenia, which is a central European country with a population of two million. IOL offers cancer genetic counselling and genetic testing of high-risk individuals at the national level and therefore serves as a referral tertiary centre for the whole country.\u003c/p\u003e \u003cp\u003eIn the Slovene HBOC cohort the prevalence and spectrum of germline \u003cem\u003ePALB2\u003c/em\u003e PV/LPVs have not yet been analysed and reported. Our study aimed to describe these PV/LPVs in \u003cem\u003ePALB2\u003c/em\u003e and analyse the types of cancer and age of cancer diagnosis in \u003cem\u003ePALB2\u003c/em\u003e PV/LPV carriers.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003eMultigene panel testing with next generation sequencing (NGS) for HBOC-related genes was introduced at the IOL in late 2014. The latest gene panel (in use since 2022) consists of nineteen genes: \u003cem\u003eATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MLH1, MSH2, MSH6, NF1, PALB2, PMS2, PTEN, RAD51C, RAD51D, STK11, TP53\u003c/em\u003e. A proband (an index case) was defined as a family member (usually an affected individual) through whom a family with a PV/LPV is ascertained. If a PV/LPV was diagnosed in at least two or more seemingly unrelated families, it was considered a recurrent variant. Our retrospective study group encompassed 5099 individuals (4564 females and 535 males) from 4610 HBOC families who underwent genetic counselling in our Cancer Genetics Clinic at the Institute of Oncology Ljubljana and were referred to germline genetic testing between January 2015 and January 2022.\u003c/p\u003e \u003cp\u003eFamily history data was collected from all tested families and all cancer diagnoses were verified in the Slovenian Cancer Registry. The registry contains data of all cancer diagnoses since 1950, when compulsory reporting of cancer diagnosis started in Slovenia. Positive family history for HBOC syndrome was defined as a family history of at least one first- or second-degree relative with breast, ovarian, prostate, or pancreatic cancer. We disregarded cases of non-melanoma skin cancer and cervical cancer in the analysis.\u003c/p\u003e \u003cp\u003eFamily members of carriers of PV/LPVs were offered either cascade genetic testing for known PV/LPV in the family or NGS panel testing in case they fulfilled the inclusion criteria for panel testing. If the tested individuals gave their consent to report secondary findings (reporting the finding of a PV/LPV not initially suspected), secondary findings were also reported according to the ACMG criteria (Richards et al. 2015). Patients who harboured variants of unknown significance were not included in the analysis.\u003c/p\u003e \u003cp\u003e All participants provided written informed consent. The present study was approved by the National Ethics Committee and the Institutional Ethics Committee of the Institute of Oncology Ljubljana (0120\u0026ndash;591/2020/3 on the 20th of January 2021). Research was conducted according to the 1975 Helsinki Declaration as revised in 1983 and the procedures used met the ethical standards of these bodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDNA Extraction\u003c/h2\u003e \u003cp\u003eDNA was isolated from blood samples according to the established laboratory protocol as previously published (16).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eNext Generation Sequencing\u003c/h2\u003e \u003cp\u003eNext generation sequencing (NGS) was performed on Illumina MiSeqDx Sequencing System using TruSight Cancer Panel or TruSight Hereditary Panel (Illumina, San Diego, CA, USA) to enrich and sequence all translated exons and \u0026plusmn;\u0026thinsp;25 bp flanking intronic regions of all HBOC panel genes. Bioinformatics and copy number analysis were performed as described by our group previously (Gornjec et al. 2019; Klančar et al. 2020). Germline variants were classified for their clinical importance according to ACMG/AMP guidelines (Plon et al. 2008; den Dunnen et al. 2016). Variants are described according to HGVS v20.05 nomenclature (den Dunnen et al. 2016). Our HBOC gene panel included 19 genes: \u003cem\u003eATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MLH1, MSH2, MSH6, NF1, PALB2, PMS2, PTEN, RAD51C, RAD51D, STK11, TP53\u003c/em\u003e. For \u003cem\u003ePALB2\u003c/em\u003e reference sequence LRG_308t1 (NM_024675.3) was used. All PV/LPV germline variants in HBOC genes detected by NGS were additionally confirmed by Multiplex Ligation-dependent Probe Amplification analysis or Sanger sequencing as described by our group previously (Stegel et al. 2022).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using SPSS software (version 25). We used descriptive statistics to describe patients\u0026rsquo; clinical, pathological, and genetic characteristics.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStudy cohort\u003c/h2\u003e \u003cp\u003eFrom January 1st 2015 to January 31st 2022, 5099 individuals (535 males and 4564 females) from 4610 families were tested for germline PV/LPVs in HBOC-related genes. The median age of individuals at the time of testing was 54 years.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePV/LPV detection rate among 4610 tested probands/families\u003c/h2\u003e \u003cp\u003eIn 19.1% (883/4610) of tested families a germline PV/LPV in HBOC-related genes was detected. \u003cem\u003eBRCA1\u003c/em\u003e PV/LPVs were detected in 8.4% (386/4610). Additionally, PV/LPVs were detected in \u003cem\u003eBRCA2\u003c/em\u003e in 4.9% (224/4610) of all probands, in \u003cem\u003eCHEK2\u003c/em\u003e in 1.8% (83/4610), in \u003cem\u003eATM\u003c/em\u003e in 1.5% (69/4610) and in \u003cem\u003ePALB2\u003c/em\u003e in 0.9% (40/4610). The frequency of all PV/LPVs in HBOC-related genes is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003ePALB2\u003c/em\u003e PV/LPVs were detected in 1.0% of all \u003cem\u003eBRCA1/2\u003c/em\u003e negative families (40/4000).\u003c/p\u003e \u003cp\u003eIn 22 out of 883 (2.5%) families, probands were diagnosed with two PV/LPVs and in one family (0.1%) one proband was diagnosed with three PV/LPVs in the HBOC-related genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePALB2 study cohort\u003c/h2\u003e \u003cp\u003eWe identified \u003cem\u003ePALB2\u003c/em\u003e PV/LPVs carriers in 40 HBOC families. In the same period, one additional family was identified, where a PV/LPV was reported as a secondary finding when a male patient with gastric cancer was tested for Lynch syndrome. Our \u003cem\u003ePALB2\u003c/em\u003e positive cohort was therefore composed of 41 families. Within these 41 families, a total of 61 family members were identified as carriers of a \u003cem\u003ePALB2\u003c/em\u003e PV/LPV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSpectrum of PALB2 PV/LPVs in the Slovenian cohort of 41 PALB2 positive families\u003c/h2\u003e \u003cp\u003eWe identified 14 different \u003cem\u003ePALB2\u003c/em\u003e PV/LPVs. \u003cem\u003ePALB2\u003c/em\u003e c.912 del p.(Val305*) had previously not been reported in the literature and was found in one proband (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFive PV/LPVs were recurrent. The two most frequent were c.509_510del and c.1451T\u0026thinsp;\u0026gt;\u0026thinsp;A, detected in 10 different families each, together encompassing almost half (20/41, 48.8%) of all PV/LPVs detected in our population.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSpectrum of \u003cem\u003ePALB2\u003c/em\u003e PV/LPVs in the Slovenian population.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePALB2\u003c/em\u003e PV/LPV type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePreviously reported\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eACMG/AMP\u003c/p\u003e \u003cp\u003eand\u003c/p\u003e \u003cp\u003eVariant class*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVariant Type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN of carriers\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN of families (% of all families)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.1451T\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003cp\u003ep.(Leu484*)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePV\u003c/p\u003e \u003cp\u003eClass 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003enonsense\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10 (24.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.509_510del\u003c/p\u003e \u003cp\u003ep.(Arg170Ilefs*14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePV\u003c/p\u003e \u003cp\u003eClass 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eframeshift\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10 (24.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.1027C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003cp\u003ep.(Gln343*)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePV\u003c/p\u003e \u003cp\u003eClass 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003enonsense\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6 (14.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.172_175del\u003c/p\u003e \u003cp\u003ep.(Gln60Argfs*7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePV\u003c/p\u003e \u003cp\u003eClass 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eframeshift\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4 (9.8%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.3549C\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003cp\u003ep.(Tyr1183*)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLPV\u003c/p\u003e \u003cp\u003eClass 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003enonsense\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2 (4.9%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.1317del\u003c/p\u003e \u003cp\u003ep.(Phe440Leufs*12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePV\u003c/p\u003e \u003cp\u003eClass 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eframeshift\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 (2.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.1240C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003cp\u003ep.(Arg414*)\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePV\u003c/p\u003e \u003cp\u003eClass 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003enonsense\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 (2.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.48G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003cp\u003ep.(Lys16=)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePV\u003c/p\u003e \u003cp\u003eClass 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003esynonymous, splicing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 (2.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.2192T\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003cp\u003ep.(Leu731*)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePV\u003c/p\u003e \u003cp\u003eClass 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003enonsense\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 (2.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.1676_1677delinsG p.(Gln559Argfs*2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePV\u003c/p\u003e \u003cp\u003eClass 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eframeshift\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 (2.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.3164dup\u003c/p\u003e \u003cp\u003ep.(Tyr1055*)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePV\u003c/p\u003e \u003cp\u003eClass 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003enonsense\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 (2.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003edeletion of exons 11\u0026ndash;12 c.(3113\u0026thinsp;+\u0026thinsp;1_3114-1)_(3350\u0026thinsp;+\u0026thinsp;1_3351-1)del\u003c/p\u003e \u003cp\u003ep.?\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePV\u003c/p\u003e \u003cp\u003eClass 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003emultiple exon deletion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 (2.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.395del\u003c/p\u003e \u003cp\u003ep.(Val132Alafs*45)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePV\u003c/p\u003e \u003cp\u003eClass 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eframeshift\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 (2.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.912del\u003c/p\u003e \u003cp\u003ep.(Val305*)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePV\u003c/p\u003e \u003cp\u003eClass 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003enonsense\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 (2.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAll\u003c/b\u003e \u003cb\u003ePALB2\u003c/b\u003e \u003cb\u003ePV/LPVs\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e61\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e41 (100%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eLegend: PV/LPV\u0026thinsp;=\u0026thinsp;pathogenic variant/likely pathogenic variant, SNV\u0026thinsp;=\u0026thinsp;single nucleotide variation, BC\u0026thinsp;=\u0026thinsp;breast cancer, OC\u0026thinsp;=\u0026thinsp;ovarian cancer, NET\u0026thinsp;=\u0026thinsp;neuroendocrine tumor, PaC\u0026thinsp;=\u0026thinsp;pancreatic cancer, f\u0026thinsp;=\u0026thinsp;female, m\u0026thinsp;=\u0026thinsp;male, ACMG/AMP classification (12) classification system proposed by Plon et al. (15).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCancer types diagnosed in PALB2\u003c/em\u003e PV/LPV \u003cem\u003ecarriers\u003c/em\u003e\u003c/p\u003e \u003cp\u003eOut of the 61 \u003cem\u003ePALB2\u003c/em\u003e-positive patients, 43 (70.5%) were diagnosed with at least one type of cancer (3 males, 40 females). The distribution of cancer types and ages at diagnosis are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The median age at the diagnosis of the first malignancy was 47 years (range 32 years \u0026ndash; 69 years).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong 36 \u003cem\u003ePALB2\u003c/em\u003e positive BC (invasive and in situ) patients, 35 were females and one was male. The median age at BC diagnosis was 46.5 years, ranging from 32 years to 69 years. The median age at genetic testing was 51 years, ranging from 21 years \u0026ndash; 79 years. Twelve different PV/LPVs were identified in \u003cem\u003ePALB2\u003c/em\u003e positive BC patients (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMale carriers with cancer\u003c/h2\u003e \u003cp\u003eEight of the \u003cem\u003ePALB2\u003c/em\u003e carriers were male and three of them were diagnosed with one malignancy each: a carrier of \u003cem\u003ePALB2\u003c/em\u003e c.1027C\u0026thinsp;\u0026gt;\u0026thinsp;T p.(Gln343*) was diagnosed with BC at the age of 69, a carrier of \u003cem\u003ePALB2\u003c/em\u003e c.3549C\u0026thinsp;\u0026gt;\u0026thinsp;G p.(Tyr1183*) was diagnosed with pancreatic cancer at 45 years of age and a carrier of \u003cem\u003ePALB2\u003c/em\u003e c.2192T\u0026thinsp;\u0026gt;\u0026thinsp;G (p.Leu731*) was diagnosed with gastric cancer at 53 years of age.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eClinical Characteristics of double heterozygotes\u003c/h2\u003e \u003cp\u003eAmong \u003cem\u003ePALB2\u003c/em\u003e PV/LPV carriers, three patients were classified as double heterozygotes (DH). Their clinical characteristics are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of double heterozygotes.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient (sex)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePALB2\u003c/em\u003e PV/LPV\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOther HBOC gene PV/LPV\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCancer type\u003c/p\u003e \u003cp\u003e(age at diagnosis)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHistopathological Characteristics\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePatient 43 (f)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003edeletion of exons 11\u0026ndash;12 c.(3113\u0026thinsp;+\u0026thinsp;1_3114-1)_(3350\u0026thinsp;+\u0026thinsp;1_3351-1)del p.?\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eATM\u003c/em\u003e\u003c/p\u003e \u003cp\u003ec.2413C\u0026thinsp;\u0026gt;\u0026thinsp;T p.(Arg805*)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBC (48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epoorly differentiated, bifocal, luminal B IDC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePatient 17 (f)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.1451T\u0026thinsp;\u0026gt;\u0026thinsp;A p.(Leu484*)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eCHEK2\u003c/em\u003e\u003c/p\u003e \u003cp\u003ec.1100del p.(Thr367Metfs*15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBC (32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epoorly differentiated, triple negative IDC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cb\u003ePatient 42 (f)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003ec.912del p.(Val305*)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cem\u003eBRCA1\u003c/em\u003e\u003c/p\u003e \u003cp\u003ec.181T\u0026thinsp;\u0026gt;\u0026thinsp;G p.(Cys61Gly)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBC (42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eatypical medullary\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOC (57)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHGSC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBC (67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eunknown histology\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePaC (69)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eadenocarcinoma\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eLegend: PV/LPV\u0026thinsp;=\u0026thinsp;pathogenic variant/likely pathogenic variant, BC\u0026thinsp;=\u0026thinsp;breast cancer, OC\u0026thinsp;=\u0026thinsp;ovarian cancer, PaC\u0026thinsp;=\u0026thinsp;pancreatic cancer, HGSC\u0026thinsp;=\u0026thinsp;high-grade serous carcinoma, FIGO\u0026thinsp;=\u0026thinsp;the International Federation of Gynecology and Obstetrics, f\u0026thinsp;=\u0026thinsp;female, m\u0026thinsp;=\u0026thinsp;male.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMultiple primary cancers\u003c/h2\u003e \u003cp\u003eIn total, 12 out of 43 (27.9%) \u003cem\u003ePALB2\u003c/em\u003e positive patients with cancer were diagnosed with more than one malignant tumor. Among them, 7/12 had negative family history and 3/7 had their first cancer diagnosed after the age of 50 years. Median interval between first diagnosis and a new primary cancer was 8 years (range 1\u0026ndash;18 years). Characteristics of patients with multiple primary malignancies are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of \u003cem\u003ePALB2\u003c/em\u003e positive patients with multiple primary malignancies in different organs.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient (sex)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePatient with PV/LPVs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eType of Cancer (age at diagnosis)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFamily history (number of affected family members)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePatient 33 (f)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePALB2\u003c/em\u003e c.1240C\u0026thinsp;\u0026gt;\u0026thinsp;T p.(Arg414*)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emelanoma (53), BC (56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003epositive (1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePatient 10 (f)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePALB2\u003c/em\u003e c.509_510del p.(Arg170Ilefs*14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epapilla Vateri (56), fallopian tube (62)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePatient 37 (f)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePALB2\u003c/em\u003e c.1676_1677delinsG p.(Gln559Argfs*2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emelanoma (47), BC (48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003epositive (1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePatient 31 (f)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePALB2\u003c/em\u003e c.172_175del p.(Gln60Argfs*7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDCIS (49), NET origo ignota (67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePatient 42 (f)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePALB2\u003c/em\u003e c.912del\u003c/p\u003e \u003cp\u003ep.(Val305*)\u003c/p\u003e \u003cp\u003e\u003cem\u003eBRCA1\u003c/em\u003e c.181T\u0026thinsp;\u0026gt;\u0026thinsp;G p.(Cys61Gly)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBC (42, 67), OC (57), PaC (69)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003epositive (1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePatient 1 (f)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePALB2\u003c/em\u003e c.509_510del p.(Arg170Ilefs*14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBC (49, 53)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003epositive (3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePatient 7 (f)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePALB2\u003c/em\u003e c.509_510del p.(Arg170Ilefs*14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBC (44, 52)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePatient 5 (f)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePALB2\u003c/em\u003e c.509_510del p.(Arg170Ilefs*14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBC (58, 62)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePatient 9 (f)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePALB2\u003c/em\u003e c.509_510del p.(Arg170Ilefs*14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBC (36, 48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePatient 18 (f)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePALB2\u003c/em\u003e c.1451T\u0026thinsp;\u0026gt;\u0026thinsp;A p.(Leu484*)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBC (52, 60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePatient 36 (f)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePALB2\u003c/em\u003e c.1317del p.(Phe440Leufs*12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBC (51), DCIS (52)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePatient 30 (f)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePALB2\u003c/em\u003e c.172_175del p.(Gln60Argfs*7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBC (40, 57)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003epositive (2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eLegend: PV/LPV\u0026thinsp;=\u0026thinsp;pathogenic variant/likely pathogenic variant, BC\u0026thinsp;=\u0026thinsp;breast cancer, OC\u0026thinsp;=\u0026thinsp;ovarian cancer, DCIS\u0026thinsp;=\u0026thinsp;ductal carcinoma in situ, NET\u0026thinsp;=\u0026thinsp;neuroendocrine tumor, PaC\u0026thinsp;=\u0026thinsp;pancreatic cancer; f - female\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, one carrier of \u003cem\u003ePALB2\u003c/em\u003e PV/LPV, who also harboured a \u003cem\u003eBRCA1\u003c/em\u003e PV/LPV, was diagnosed with four primary cancers: twice with BC (at 42 and 67 years), a high-grade serous carcinoma of the ovary (age at diagnosis 57) and a pancreatic adenocarcinoma (age at diagnosis 69). Additionally, two \u003cem\u003ePALB2\u003c/em\u003e positive patients both developed BC (aged 48 and 56, respectively) and melanoma (aged 47 and 53, respectively).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePALB2 positive carriers without cancer diagnosis\u003c/h2\u003e \u003cp\u003eOur cohort of \u003cem\u003ePALB2\u003c/em\u003e PV/LPV carriers without a cancer diagnosis consisted of 18 individuals from 11 families. Thirteen were female and five were male. Two of them were identified through panel testing and 16 by cascade testing. Median age at genetic testing was 44 years (range 21 \u0026ndash; to 75 years).\u003c/p\u003e \u003cp\u003eAmong these individuals, \u003cem\u003ePALB2\u003c/em\u003e c.1451T\u0026thinsp;\u0026gt;\u0026thinsp;A p.(Leu484*) PV/LPV was found in 11 individuals from six families, \u003cem\u003ePALB2\u003c/em\u003e c.509_510del (p.(Arg170Ilefs14)) was found in two individuals from two different families, \u003cem\u003ePALB2\u003c/em\u003e c.1317del p.(Phe440Leufs12) was found in two individuals from the same family, and one individual was identified with each of the following PV/LPVs: \u003cem\u003ePALB2\u003c/em\u003e c.1240C\u0026thinsp;\u0026gt;\u0026thinsp;T p.(Arg414*), \u003cem\u003ePALB2\u003c/em\u003e c.1027C\u0026thinsp;\u0026gt;\u0026thinsp;T p.(Gln343*), and \u003cem\u003ePALB2\u003c/em\u003e c.172_175del p.(Gln60Argfs*7).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHaving epidemiological data on the frequency and spectrum of germline PV/LPVs associated with different hereditary cancers is of the utmost importance for every country aiming to organize an optimal cancer prevention programme and optimize cancer patients\u0026rsquo; management. In the Slovene population, data on the occurrence of germline PV/LPVs in \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e genes and the clinicopathological characteristics of malignancies in those patients, have already been reported (20). Additionally, the spectrum of \u003cem\u003eBRCA1\u003c/em\u003e and \u003cem\u003eBRCA2\u003c/em\u003e PV/LPVs in male breast cancer patients, and characteristics of breast cancer patients with \u003cem\u003eCHEK2\u003c/em\u003e PV/LPVs have been studied (Besic et al. 2008; Krajc et al. 2014; Cvelbar et al. 2017; Nizic-Kos et al. 2021). \u003cem\u003ePALB2\u003c/em\u003e is one of the most common HBOC-related genes, found in approximately 1% of \u003cem\u003eBRCA1/\u003c/em\u003e2 negative BC patients (Wu et al. 2020; Woodward et al. 2021) and is highly penetrant (Yang et al. 2020b). Nevertheless, very little is known about the characteristics of carriers of \u003cem\u003ePALB2\u003c/em\u003e PV/LPVs and the clinicopathological characteristics of tumors diagnosed in these patients. This gap in knowledge underscores the importance of further research into PALB2-associated cancers, not only for specialized institutions but also for primary care physicians.\u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDetection rate and spectrum of PALB2 PV/LPVs\u003c/h2\u003e \u003cp\u003eAmong all families tested with HBOC panel, we identified at least one germline PV/LPV in one of the genes in 19.1%. \u003cem\u003ePALB2\u003c/em\u003e was the fifth most commonly mutated HBOC-related gene (following \u003cem\u003eBRCA1, BRCA2, CHEK2\u003c/em\u003e and \u003cem\u003eATM\u003c/em\u003e). PV/LPVs in \u003cem\u003ePALB2\u003c/em\u003e represent 0.9% of all individuals tested, which was expected as it had previously been reported that 0.2\u0026ndash;0.9% of women with breast cancer who undergo genetic testing will carry germline PV/LPV in \u003cem\u003ePALB2\u003c/em\u003e (Hu et al. 2021).\u003c/p\u003e \u003cp\u003eIn our cohort the frequency of PV/LPVs in more than one gene from the HBOC panel in tested individuals is 2.6% (23/883) and is higher than the one reported in the literature, where we can find data ranging from 0.5 to 1% (Rosenthal et al. 2017; Shao et al. 2020). That may be due to the high prevalence of recurrent PV/LPVs in our population (Krajc et al. 2008; Stegel et al. 2011; Krajc et al. 2014).\u003c/p\u003e \u003cp\u003eIn 41 families 14 different \u003cem\u003ePALB2\u003c/em\u003e PV/LPVs were detected. \u003cem\u003ePALB2\u003c/em\u003e c.912 del p.(Val305*) had not been reported previously and was found in one proband. Newly described \u003cem\u003ePALB2\u003c/em\u003e PV/LPVs in patients with BC are important since they can contribute to international databases and patients may benefit from prevention and treatment options.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eRecurrent PV/LPVs in PALB2 in the Slovenian population\u003c/h2\u003e \u003cp\u003eFive PV/LPVs in \u003cem\u003ePALB2\u003c/em\u003e were recurrent (diagnosed in at least two or more seemingly unrelated families) in our population, with \u003cem\u003ePALB2\u003c/em\u003e c.509_510del and \u003cem\u003ePALB2\u003c/em\u003e c.1451T\u0026thinsp;\u0026gt;\u0026thinsp;A being the two most frequent, detected in 10 different families each, and together encompassing almost half (20/41 or 48.8%) of all detected PV/LPVs in our population. Recurrent \u003cem\u003ePALB2\u003c/em\u003e PV/LPVs have been reported in other populations as well, such as those from Argentina (Gonzalez et al. 2022), Finland (Erkko et al. 2007) and Poland (Noskowicz et al. 2014). \u003cem\u003ePALB2\u003c/em\u003e c.509_510del has been described by Noskowitz et al. as being present in about 1 in 400 unselected breast cancer patients from Central Europe (Germany) and Eastern Europe (Belarus, Russia) (Noskowicz et al. 2014). \u003cem\u003ePALB2\u003c/em\u003e c.509_510del has also been described as a recurrent mutation in BC and OC patients from Poland (Kluska et al. 2017). We found no reports on the presence of \u003cem\u003ePALB2\u003c/em\u003e c.509_510del in Western European, Asian or American populations. While the Slovenian language does belong to the South Slavic language group, genetic studies have revealed close genetic affiliations with West Slavic populations, such as Poles, suggesting a common Slavic ancestor originating from the Dnieper basin (Zupan et al. 2013). We found no genotype-phenotype correlation studies for this specific PV. Of note, three patients from our cohort (33.3%) were diagnosed with a metachronous contralateral BC, one additional (11.1%) with a metachronous contralateral and ipsilateral BC. Additionally, we found two cases of ovarian cancer (10.5% of \u003cem\u003ePALB2\u003c/em\u003e c.509_510delGA carriers) with a median age at diagnosis 56.5 years (range 51 years \u0026ndash; 62 years), one case of carcinoma of the papilla Vateri and one malignant melanoma. Based on our data BC patients harbouring \u003cem\u003ePALB2\u003c/em\u003e c.509_510del are at high risk of developing a second BC and OC, making them high-risk group among \u003cem\u003ePALB2\u003c/em\u003e PV carriers.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePALB2\u003c/em\u003e c.1451T\u0026thinsp;\u0026gt;\u0026thinsp;A has not yet been described as a recurrent mutation in any population in the literature, however it has been found in nineteen individuals from ten different families in our cohort, which makes it a unique recurrent mutation in the Slovenian population. Among nineteen carriers of the above-mentioned PV, eight were diagnosed with breast cancer, with a median age at diagnosis 49 years. While it is challenging to establish genotype-phenotype correlation, this information could still be valuable in everyday clinical practice and may be included in the studies with bigger sample sizes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMalignancies among PALB2 PV/LPV carriers\u003c/h2\u003e \u003cp\u003eIt has been known for more than a decade that \u003cem\u003ePALB2\u003c/em\u003e PV/LPVs increase BC risk (Hamdan and Nowak 2022). The risk is 2\u0026ndash;30 times higher than in the general population, depending on the type of PV/LPV, age, and family history and \u003cem\u003ePALB2\u003c/em\u003e is considered a high-penetrance susceptibility gene for BC (Antoniou et al. 2014). As expected, the most common malignancy diagnosed in our cohort was BC in 36 patients, which confirms this association. Recent research has strengthened the correlation between \u003cem\u003ePALB2\u003c/em\u003e PV/LPVs and ovarian cancer. Yang et al. demonstrated a substantial association between germline \u003cem\u003ePALB2\u003c/em\u003e PV/LPVs and ovarian cancer with a risk ratio of around 3 and the lifetime risk of OC estimated to be around 3\u0026ndash;5%(Yang et al. 2020a). In our cohort, four patients were diagnosed with a high-grade serous carcinoma of the ovary or the fallopian tube and one with a borderline ovarian carcinoma. There were no cases of primary peritoneal serous carcinoma. OC was the second most prevalent malignancy in our study. Two \u003cem\u003ePALB2\u003c/em\u003e PV/LPV carriers in our cohort (2/61, 3.3%) were diagnosed with PaC and additional carrier with carcinoma of Papilla Vateri. Indeed there is emerging evidence that \u003cem\u003ePALB2\u003c/em\u003e PV/LPVs predispose patients to pancreatic cancer. It is estimated that 3\u0026ndash;4% of patients with famillial PaC are expected to harbour \u003cem\u003ePALB2\u003c/em\u003e PV/LPV. In the newest version of the National Comprehensive Cancer Network (NCCN) Guidelines enhanced screening not only for BC (with possible risk reducing surgeries), but also for OC (risk reducing surgery is offered as an option) and PaC is recommended (Clinical et al. 2024). Additionally, we have identified 3 cases of malignant melanoma, 1 NET and 1 gastric cancer in our cohort, there is, however, insufficient evidence to draw a conclusion about these types of malignancies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eDouble heterozygotes\u003c/h2\u003e \u003cp\u003eThe increased use of multigene panels in the recent years has led to identification of individuals harbouring more than one PV/LPV in cancer susceptibility genes, although the data is still scarce. Double heterozygous PV in \u003cem\u003eBRCA1/2\u003c/em\u003e are identified in 0.3% of Ashkenazi and are very rare in other populations. Lavie et al. suggest that DH PV in \u003cem\u003eBRCA1/2\u003c/em\u003e in females of Ashkenazi Jewish heritage does not seem to cause a more severe phenotype than in cases where only one of the genes is implicated (Lavie et al. 2011). The results of the studies on DH VP in HBOC-related genes in other populations have been conflicting and inconclusive. To the best of our knowledge no research has been conducted on DH with one of the variants being \u003cem\u003ePALB2\u003c/em\u003e PV/LPV. A case report by Agiannitopoulos described a female patient, who was diagnosed at the age of 42 with endometrial cancer. DH in \u003cem\u003ePALB2\u003c/em\u003e and \u003cem\u003eMSH\u003c/em\u003e explained the remarkable family history of ovarian, breast, kidney and colorectal cancer and consequently the surveillance of family members was adjusted (Agiannitopoulos et al. 2020). There is emerging evidence of multiplicative effect of presence of PV/LPVs in more than one cancer susceptibility gene. Heidemann et al. showed that Caucasian female DH for \u003cem\u003eBRCA1/2\u003c/em\u003e seem to develop BC at a younger age and have more severe disease than carriers of a single \u003cem\u003eBRCA1/2\u003c/em\u003e PV/LPV (Heidemann et al. 2012). Similarly, Sokolenko et al. pointed out that the presence of additional gene defect in female \u003cem\u003eBRCA1\u003c/em\u003e PV carriers may further increase their chances for cancer (Sokolenko et al. 2014). We have identified three DH carriers in our \u003cem\u003ePALB2\u003c/em\u003e cohort with additional PV/LPVs in \u003cem\u003eATM\u003c/em\u003e, \u003cem\u003eCHEK2\u003c/em\u003e, and \u003cem\u003eBRCA1\u003c/em\u003e. The clinical presentations varied. DH patients with PV/LPVs in \u003cem\u003ePALB2\u003c/em\u003e and \u003cem\u003eATM\u003c/em\u003e or \u003cem\u003eCHEK2\u003c/em\u003e were diagnosed with BC at 48 and 32 years, respectively. Patient who was found to harbour \u003cem\u003ePALB2\u003c/em\u003e and \u003cem\u003eBRCA1\u003c/em\u003e PV/LPV was diagnosed with 4 malignancies: two BC, OC and PaC, supporting the multiplying effect of DH. Genetic counselling for DH carriers is complex and further studies are required to elucidate its biological effect.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStrengths and limitations of our study\u003c/h2\u003e \u003cp\u003eThere are several limitations of our study. The expected population burden of \u003cem\u003ePALB2\u003c/em\u003e PV/LPVs in the Slovenian population is 0.13% (Kotnik et al. 2023). The absolute number of included PV/LPV carriers was small (61). 43 (70.5%) had a cancer diagnosis. However, our cohort represents 2% of the expected population of \u003cem\u003ePALB2\u003c/em\u003e PV/LPV carriers in Slovenia. Also, in comparison to the literature, where mostly case series are described, this is a large cohort of \u003cem\u003ePALB2\u003c/em\u003e PV/LPV carriers. The subgroups of patients with different PV/LPV were very small, therefore we were not able to analyse them separately. The retrospective collection of data is always unfavourable, since data can be missing or inappropriately understood, however as it can be seen from our data, the information regarding patients\u0026rsquo; and tumours\u0026rsquo; characteristics was complete for patients in our study.\u003c/p\u003e \u003cp\u003eIts strength lies in a reliable family history, with information obtained from the Slovenian National Cancer Registry. It is one of the oldest Registries in Europe, where the diagnoses are cross-checked with histopathological reports.\u003c/p\u003e \u003cp\u003eIn cases of rare genetic diseases large multicentric studies are required to achieve a substantial number of patients and we hope these will be able to benefit from our cohort of \u003cem\u003ePALB2\u003c/em\u003e PV/LPV carriers.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis report provides the first comprehensive insight into the genotype and phenotype of \u003cem\u003ePALB2\u003c/em\u003e PV/LPV carriers in Slovenia. The frequency of \u003cem\u003ePALB2\u003c/em\u003e PV/LPV in Slovenia is consistent with rates reported in other countries, accounting for 0.9% of all individuals tested for PVs in HBOC-related genes. Notably, we identified two recurrent \u003cem\u003ePALB2\u003c/em\u003e PV/LPVs within our population, collectively encompassing nearly half of all affected families. Of particular interest is the \u003cem\u003ePALB2\u003c/em\u003e c.1451T\u0026thinsp;\u0026gt;\u0026thinsp;A variant, which has not been previously documented as a recurrent mutation in any population, rendering it unique to Slovenia The most common malignancy in our cohort was as expected BC, followed by OC and PaC, which adds to the existing evidence of \u003cem\u003ePALB2\u003c/em\u003e involvement in the pathogenesis of thesecancer types. Despite the rarity of \u003cem\u003ePALB2\u003c/em\u003e carriers, who also carry PV/LPVs in other HBOC-related genes, we have identified three such individuals, and studying their disease characteristic can help elucidate the biological effect of being a DH. Overall, the results of our study provide valuable genotype and phenotype data from \u003cem\u003ePALB2\u003c/em\u003e positive patients which may already be utilized in a population specific assessment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe study was supported by the Slovenian Research Agency, program number P3-0289.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting Interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthor Contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Vita Andreja Mesarič, Mateja Krajc, Methodology: Mateja Krajc, Kristina Drusany Starič, Simona Hotujec; Formal analysis and investigation: Vita Drago\u0026scaron; \u0026Scaron;etrajčič, Petra \u0026Scaron;kerl, Srdjan Novaković, Vida Stegel; Writing - original draft preparation: Vita Andreja Mesarič; Writing - review and editing: Mateja Krajc, Ana Blatnik, Ksenija Strojnik, Kristina Drusany Starič. \u003cem\u003eAll authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eData availability\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEthics approval\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll participants provided written informed consent. The present study was approved by the National Ethics Committee and the Institutional Ethics Committee of the Institute of Oncology Ljubljana (0120-591/2020/3 on the 20\u003csup\u003eth\u003c/sup\u003e of January 2021). Research was conducted according to the 1975 Helsinki Declaration as revised in 1983 and the procedures used met the ethical standards of these bodies.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAgiannitopoulos K, Papadopoulou E, Tsaousis GN, Pepe G, Kampouri S, Patsea E, Lypas G, Nasioulas G (2020) Report of a germline double heterozygote in MSH2 and PALB2. 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BMC Med Genet 12:1\u0026ndash;11. https://doi.org/10.1186/1471-2350-12-9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalsh CS (2015) Two decades beyond BRCA1/2: Homologous recombination, hereditary cancer risk and a target for ovarian cancer therapy? Gynecol Oncol 137:343\u0026ndash;350\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWoodward ER, van Veen EM, Forde C, Harkness EF, Byers HJ, Ellingford JM, Burghel GJ, Schlech H, Bowers NL, Wallace AJ, Howell SJ, Howell A, Lalloo F, Newman WG, Smith MJ, Gareth Evans D (2021) Clinical utility of testing for PALB2 and CHEK2 c.1100delC in breast and ovarian cancer. Genetics in Medicine 23:1969\u0026ndash;1976. https://doi.org/10.1038/s41436-021-01234-6\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu Y, Ouyang T, Li J, Wang T, Fan Z, Fan T, Lin B, Xu Y, Xie Y (2020) Spectrum and clinical relevance of PALB2 germline mutations in 7657 Chinese BRCA1/2-negative breast cancer patients. Breast Cancer Res Treat 179:605\u0026ndash;614. https://doi.org/10.1007/s10549-019-05483-7\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang X, Leslie G, Doroszuk A, Schneider S, Allen J, Decker B, Dunning AM, Redman J, Scarth J, Plaskocinska I, Luccarini C, Shah M, Pooley K, Dorling L, Leei A, Adank MA, Adlard J, Aittom\u0026auml;ki K, Andrulis IL, Ang P, Barwell J, Bernstein JL, Bobolis K, Borg \u0026Aring;, Blomqvist C, Claes KBM, Concannon P, Cuggia A, Culver JO, Damiola F, De Pauw A, Diez O, Dolinsky JS, Domchek SM, Engel C, Evans DG, Fostira F, Garber J, Golmard L, Goode EL, Gruber SB, Hahnen E, Hake C, Heikkinen T, Hurley JE, Janavicius R, Kleibl Z, Kleiblova P, Konstantopoulou I, Kvist A, Laduca H, Lee ASG, Lesueur F, Maher ER, Mannermaa A, Manoukian S, McFarland R, McKinnon W, Meindl A, Metcalfe K, Taib NAM, Moilanen J, Nathanson KL, Neuhausen S, Ng PS, Nguyen-Dumont T, Nielsen SM, Obermair F, Offit K, Olopade OI, Ottini L, Penkert J, Pylk\u0026auml;s K, Radice P, Ramus SJ, Rudaitis V, Side L, Silva-Smith R, Silvestri V, Skytte AB, Slavin T, Soukupova J, Tondini C, Trainer AH, Unzeitig G, Usha L, Van Overeem Hansen T, Whitworth J, Wood M, Yip CH, Yoon SY, Yussuf A, Zogopoulos G, Goldgar D, Hopper JL, Chenevix-Trench G, Pharoah P, George SHL, Balma\u0026ntilde;a J, Houdayer C, James P, El-Haffaf Z, Ehrencrona H, Janatova M, Peterlongo P, Nevanlinna H, Schmutzler R, Teo SH, Robson M, Pal T, Couch F, Weitzel JN, Elliott A, Southey M, Winqvist R, Easton DF, Foulkes WD, Antoniou AC, Tischkowitz M (2020a) Cancer risks associated with germline PALB2 pathogenic variants: An international study of 524 families. Journal of Clinical Oncology 38:674\u0026ndash;685\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang X, Leslie G, Doroszuk A, Schneider S, Allen J, Decker B, Dunning AM, Redman J, Scarth J, Plaskocinska I, Luccarini C, Shah M, Pooley K, Dorling L, Leei A, Adank MA, Adlard J, Aittom\u0026auml;ki K, Andrulis IL, Ang P, Barwell J, Bernstein JL, Bobolis K, Borg \u0026Aring;, Blomqvist C, Claes KBM, Concannon P, Cuggia A, Culver JO, Damiola F, De Pauw A, Diez O, Dolinsky JS, Domchek SM, Engel C, Evans DG, Fostira F, Garber J, Golmard L, Goode EL, Gruber SB, Hahnen E, Hake C, Heikkinen T, Hurley JE, Janavicius R, Kleibl Z, Kleiblova P, Konstantopoulou I, Kvist A, Laduca H, Lee ASG, Lesueur F, Maher ER, Mannermaa A, Manoukian S, McFarland R, McKinnon W, Meindl A, Metcalfe K, Taib NAM, Moilanen J, Nathanson KL, Neuhausen S, Ng PS, Nguyen-Dumont T, Nielsen SM, Obermair F, Offit K, Olopade OI, Ottini L, Penkert J, Pylk\u0026auml;s K, Radice P, Ramus SJ, Rudaitis V, Side L, Silva-Smith R, Silvestri V, Skytte AB, Slavin T, Soukupova J, Tondini C, Trainer AH, Unzeitig G, Usha L, Van Overeem Hansen T, Whitworth J, Wood M, Yip CH, Yoon SY, Yussuf A, Zogopoulos G, Goldgar D, Hopper JL, Chenevix-Trench G, Pharoah P, George SHL, Balma\u0026ntilde;a J, Houdayer C, James P, El-Haffaf Z, Ehrencrona H, Janatova M, Peterlongo P, Nevanlinna H, Schmutzler R, Teo SH, Robson M, Pal T, Couch F, Weitzel JN, Elliott A, Southey M, Winqvist R, Easton DF, Foulkes WD, Antoniou AC, Tischkowitz M (2020b) Cancer risks associated with germline PALB2 pathogenic variants: An international study of 524 families. Journal of Clinical Oncology 38:674\u0026ndash;685. https://doi.org/10.1200/JCO.19.01907\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZupan A, Vrabec K, Glavač D (2013) The paternal perspective of the slovenian population and its relationship with other populations. Ann Hum Biol 40:515\u0026ndash;526. https://doi.org/10.3109/03014460.2013.813584\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"human-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hugm","sideBox":"Learn more about [Human Genomics](http://humgenomics.biomedcentral.com/)","snPcode":"40246","submissionUrl":"https://submission.nature.com/new-submission/40246/3","title":"Human Genomics","twitterHandle":"@OAgenetics","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"HBOC, PALB2, breast cancer, ovarian cancer, risk-reducing salpingoophorectomy, recurrent pathogenic variants","lastPublishedDoi":"10.21203/rs.3.rs-4569442/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4569442/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eIntroduction\u003c/em\u003e: The prevalence and spectrum of \u003cem\u003ePALB2\u003c/em\u003e pathogenic/likely pathogenic variants (PV/LPVs) may vary across different regions, and these have not yet been analysed and reported in Slovenian HBOC families.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMethods\u003c/em\u003e: We performed a retrospective analysis of all 5099 consecutively tested individuals from 4610 families who fulfilled national criteria for HBOC-panel testing from January 2015 to January 2022. After genetic counselling, genetic testing with next generation sequencing was performed for all probands and cascade testing was offered to their blood relatives.\u003c/p\u003e \u003cp\u003e \u003cem\u003eResults\u003c/em\u003e: Among all probands tested 0.9% (40/4610) were \u003cem\u003ePALB2\u003c/em\u003e PV/LPV carriers. 14 different \u003cem\u003ePALB2\u003c/em\u003e PV/LPVs were detected, one of them was novel. Five PV/LPVs were found to be recurrent in Slovenian population with two most frequent being c.509_510del and c.1451T\u0026thinsp;\u0026gt;\u0026thinsp;A. Altogether, 61 individuals from 41 \u003cem\u003ePALB2\u003c/em\u003e positive families were identified, 43 being cancer patients. 27.9% \u003cem\u003ePALB2\u003c/em\u003e-positive cancer patients were diagnosed with more than one malignant tumour. We identified three double heterozygote carriers with additional PV/LPVs in \u003cem\u003eATM, CHEK2\u003c/em\u003e and \u003cem\u003eBRCA1.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eDiscussion\u003c/em\u003e: This report provides the first comprehensive description of molecular and clinical characteristics of \u003cem\u003ePALB2\u003c/em\u003e carriers in Slovenia. The frequency of \u003cem\u003ePALB2\u003c/em\u003e pathogenic variants in the Slovenian HBOC accounts for 0.9% of all individuals tested for PVs in HBOC-related genes. Our study adds a novel recurrent mutation, which is unique to the Slovenian context and one PV/LPVs, which had not been reported in the literature so far. The results of our study add information on genotype and phenotype in \u003cem\u003ePALB2-\u003c/em\u003epositive patients and may be used for population specific assessment.\u003c/p\u003e \u003cp\u003e\u003cb\u003eEthics approval\u003c/b\u003e: The present study was approved by the National Ethics Committee and the Institutional Ethics Committee of the Institute of Oncology Ljubljana (0120\u0026ndash;591/2020/3 on the 20th of January 2021).\u003c/p\u003e","manuscriptTitle":"Two recurrent pathogenic/likely pathogenic variants in PALB2 account for almost half of PALB2 positive families in Slovenia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-08 17:09:04","doi":"10.21203/rs.3.rs-4569442/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-15T13:06:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-14T16:09:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-14T10:54:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-04T14:39:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"181585060199015178610439497446107537683","date":"2024-06-13T08:49:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"19444685424255445032756820147595082253","date":"2024-06-13T08:36:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"175077068816691100070189816088882897162","date":"2024-06-13T08:25:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-13T08:14:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-12T23:29:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-12T23:28:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Human Genomics","date":"2024-06-12T10:17:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"human-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hugm","sideBox":"Learn more about [Human Genomics](http://humgenomics.biomedcentral.com/)","snPcode":"40246","submissionUrl":"https://submission.nature.com/new-submission/40246/3","title":"Human Genomics","twitterHandle":"@OAgenetics","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a402fc7a-36c4-48ac-a237-b35de3c9f2de","owner":[],"postedDate":"July 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-23T16:05:57+00:00","versionOfRecord":{"articleIdentity":"rs-4569442","link":"https://doi.org/10.1186/s40246-024-00706-5","journal":{"identity":"human-genomics","isVorOnly":false,"title":"Human Genomics"},"publishedOn":"2024-12-18 15:58:35","publishedOnDateReadable":"December 18th, 2024"},"versionCreatedAt":"2024-07-08 17:09:04","video":"","vorDoi":"10.1186/s40246-024-00706-5","vorDoiUrl":"https://doi.org/10.1186/s40246-024-00706-5","workflowStages":[]},"version":"v1","identity":"rs-4569442","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4569442","identity":"rs-4569442","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}