Characterization of HER2-low breast cancer in young women with germline BRCA1/2 pathogenetic variants: Results of a large international retrospective cohort study.

BACKGROUND: Breast cancer (BC) in women aged ≤40 years carrying germline pathogenetic variants (PVs) in BRCA1/2 genes is infrequent but often associated with aggressive features. Human epidermal growth factor receptor 2 (HER2)-low-expressing BC has recently emerged as a novel therapeutic target but has not been characterized in this rare patient subset. METHODS: Women aged ≤40 years with newly diagnosed early-stage HER2-negative BC (HER2-0 and HER2-low) and germline BRCA1/2 PVs from 78 health care centers worldwide were retrospectively included. Chi-square test and Student t-test were used to describe variable distribution between HER2-0 and HER2-low. Associations with HER2-low status were assessed with logistic regression. Kaplan-Meier method and Cox regression analysis were used to assess disease-free survival (DFS) and overall survival. Statistical significance was considered for p ≤ .05. RESULTS: Of 3547 included patients, 32.3% had HER2-low BC, representing 46.3% of hormone receptor-positive and 21.3% of triple-negative (TN) tumors. HER2-low vs. HER2-0 BC were more often of grade 1/2 (p < .001), hormone receptor-positive (p < .001), and node-positive (p = .003). BRCA2 PVs were more often associated with HER2-low than BRCA1 PVs (p < .001). HER2-low versus HER2-0 showed better DFS (hazard ratio [HR], 0.86; 95% CI, 0.76-0.97) in the overall population and more favorable DFS (HR, 0.78; 95% CI, 0.64-0.95) and overall survival (HR, 0.65; 95% CI, 0.46-0.93) in the TN subgroup. Luminal A-like tumors in HER2-low (p = .014) and TN and luminal A-like in HER2-0 (p = .019) showed the worst DFS. CONCLUSIONS: In young patients with HER2-negative BC and germline BRCA1/2 PVs, HER2-low disease was less frequent than expected and more frequently linked to BRCA2 PVs and associated with luminal-like disease. HER2-low status was associated with a modestly improved prognosis.

A Validated Highly Sensitive Microsatellite Instability Assay Accurately Identifies Individuals Harboring Biallelic Germline PMS2 Pathogenic Variants in Constitutional Mismatch Repair Deficiency.

BACKGROUND: Constitutional mismatch repair deficiency (CMMRD) is a rare and extraordinarily penetrant childhood-onset cancer predisposition syndrome. Genetic diagnosis is often hampered by the identification of mismatch repair (MMR) variants of unknown significance and difficulties in PMS2 analysis, the most frequently mutated gene in CMMRD. We present the validation of a robust functional tool for CMMRD diagnosis and the characterization of microsatellite instability (MSI) patterns in blood and tumors. METHODS: The highly sensitive assessment of MSI (hs-MSI) was tested on a blinded cohort of 66 blood samples and 24 CMMRD tumor samples. Hs-MSI scores were compared with low-pass genomic instability scores (LOGIC/MMRDness). The correlation of hs-MSI scores in blood with age of cancer onset and the distribution of insertion-deletion (indel) variants in microsatellites were analyzed in a series of 169 individuals (n = 68 CMMRD, n = 124 non-CMMRD). RESULTS: Hs-MSI achieved high accuracy in the identification of CMMRD in blood (sensitivity 98.5% and specificity 100%) and detected MSI in CMMRD-associated tumors. Hs-MSI had a strong positive correlation with whole low-pass genomic instability LOGIC scores (r = 0.89, P = 2.2e-15 in blood and r = 0.82, P = 7e-3 in tumors). Indel distribution identified PMS2 pathogenic variant (PV) carriers from other biallelic MMR gene PV carriers with an accuracy of 0.997. Higher hs-MSI scores correlated with younger age at diagnosis of the first tumor (r = -0.43, P = 0.011). CONCLUSIONS: Our study confirms the accuracy of the hs-MSI assay as ancillary testing for CMMRD diagnosis, which can also characterize MSI patterns in CMMRD-associated cancers. Hs-MSI is a powerful tool to pinpoint PMS2 as the affected germline gene and thus potentially personalize cancer risk.

Clinical and psychological implications of secondary and incidental findings in cancer susceptibility genes after exome sequencing in patients with rare disorders.

BACKGROUND/OBJECTIVES: Exome sequencing may identify pathogenic variants unrelated with the purpose of the analysis. We investigated the frequency of secondary and incidental findings (SF/IF) in cancer susceptibility genes (CSG), their clinical actionability and the psychological impact in individuals with an SF/IF (cases) compared with individuals tested due to their cancer history (controls). METHODS: This study analysed 533 exomes ordered for non-cancer conditions. Medical records were reviewed for clinical actionability of SF/IF. Psychological impact was analysed using the Multidimensional Impact of Cancer Risk Assessment (MICRA) scale and compared between cases and controls with a propensity score weighting method. RESULTS: The frequency of SF/IF in CSG was 2.1% (95% CI 1.1% to 3.8%): three BRCA2, three PMS2, two SDHB, and one each in BRCA1, MLH1 and RAD51C. Among the relatives, 18 were carriers. Twenty enrolled for surveillance, and a neoplasm was diagnosed in 20%: three paragangliomas and one breast cancer. Cases presented higher MICRA mean scores than controls (21.3 vs 16.2 in MICRA total score, 6.3 vs 4.2 in the distress subscale, and 8.3 vs 6.6 in the uncertainty subscale; all p<0.001). CONCLUSION: SF/IF in CSG were identified in 2.1% of patients. Despite a numerically higher psychological impact, the identification of SF/IF allowed early detection and cancer prevention in families without cancer history.

Pregnancy After Breast Cancer in Young BRCA Carriers: An International Hospital-Based Cohort Study.

Importance: Young women with breast cancer who have germline pathogenic variants in BRCA1 or BRCA2 face unique challenges regarding fertility. Previous studies demonstrating the feasibility and safety of pregnancy in breast cancer survivors included limited data regarding BRCA carriers. Objective: To investigate cumulative incidence of pregnancy and disease-free survival in young women who are BRCA carriers. Design, Setting, and Participants: International, multicenter, hospital-based, retrospective cohort study conducted at 78 participating centers worldwide. The study included female participants diagnosed with invasive breast cancer at age 40 years or younger between January 2000 and December 2020 carrying germline pathogenic variants in BRCA1 and/or BRCA2. Last delivery was October 7, 2022; last follow-up was February 20, 2023. Exposure: Pregnancy after breast cancer. Main Outcomes and Measures: Primary end points were cumulative incidence of pregnancy after breast cancer and disease-free survival. Secondary end points were breast cancer-specific survival, overall survival, pregnancy, and fetal and obstetric outcomes. Results: Of 4732 BRCA carriers included, 659 had at least 1 pregnancy after breast cancer and 4073 did not. Median age at diagnosis in the overall cohort was 35 years (IQR, 31-38 years). Cumulative incidence of pregnancy at 10 years was 22% (95% CI, 21%-24%), with a median time from breast cancer diagnosis to conception of 3.5 years (IQR, 2.2-5.3 years). Among the 659 patients who had a pregnancy, 45 (6.9%) and 63 (9.7%) had an induced abortion or a miscarriage, respectively. Of the 517 patients (79.7%) with a completed pregnancy, 406 (91.0%) delivered at term (≥37 weeks) and 54 (10.4%) had twins. Among the 470 infants born with known information on pregnancy complications, 4 (0.9%) had documented congenital anomalies. Median follow-up was 7.8 years (IQR, 4.5-12.6 years). No significant difference in disease-free survival was observed between patients with or without a pregnancy after breast cancer (adjusted hazard ratio, 0.99; 95% CI, 0.81-1.20). Patients who had a pregnancy had significantly better breast cancer-specific survival and overall survival. Conclusions and Relevance: In this global study, 1 in 5 young BRCA carriers conceived within 10 years after breast cancer diagnosis. Pregnancy following breast cancer in BRCA carriers was not associated with decreased disease-free survival. Trial Registration: ClinicalTrials.gov Identifier: NCT03673306.

Role of psychological background in cancer susceptibility genetic testing distress: It is not only about a positive result.

Clinical and familial factors predict psychological distress after genetic testing for cancer susceptibility. However, the contribution of an individual's psychological background to such distress is unclear. This study aims to analyze the psychological impact of genetic testing and to identify the profile of individuals at higher risk. This is a longitudinal multicenter study of individuals undergoing genetic testing for cancer susceptibility. Demographic, clinical, genetic, familial, and psychological (personality types, cancer worry) characteristics were assessed by validated questionnaires the day of genetic testing. Distress, uncertainty, and positive experience perception (MICRA scale) were evaluated at the results disclosure visit, and 3 and 12 months afterwards. Multivariate analysis was performed. A total of 714 individuals were included. A high neuroticism score, high baseline cancer worry, and a positive genetic test result were independently associated with higher psychological impact (p-value < 0.05). The highest risk profile (10% of the cohort) included women with high level of neuroticism and a positive result. Uncertainty was mainly associated with a high level of neuroticism, regardless of the genetic test result. A holistic approach to personalized germline genetic counseling should include the assessment of personality dimensions.

Patients' and professionals' perspective of non-in-person visits in hereditary cancer: predictors and impact of the COVID-19 pandemic.

PURPOSE: To identify predictors of patient acceptance of non-in-person cancer genetic visits before and after the COVID-19 pandemic and assess the preferences of health-care professionals. METHODS: Prospective multicenter cohort study (N = 578, 1 February 2018-20 April 2019) and recontacted during the COVID-19 lockdown in April 2020. Health-care professionals participated in May 2020. Association of personality traits and clinical factors with acceptance was assessed with multivariate analysis. RESULTS: Before COVID-19, videoconference was more accepted than telephone-based visits (28% vs. 16% pretest, 30% vs. 19% post-test). Predictors for telephone visits were age (pretest, odds ratio [OR] 10-year increment = 0.79; post-test OR 10Y = 0.78); disclosure of panel testing (OR = 0.60), positive results (OR = 0.52), low conscientiousness group (OR = 2.87), and post-test level of uncertainty (OR = 0.93). Predictors for videoconference were age (pretest, OR 10Y = 0.73; post-test, OR 10Y = 0.75), educational level (pretest: OR = 1.61), low neuroticism (pretest, OR = 1.72), and post-test level of uncertainty (OR = 0.96). Patients' reported acceptance for non-in-person visits after COVID-19 increased to 92% for the pretest and 85% for the post-test. Health-care professionals only preferred non-in-person visits for disclosure of negative results (83%). CONCLUSION: These new delivery models need to recognize challenges associated with age and the psychological characteristics of the population and embrace health-care professionals' preferences.

Natural gene therapy by reverse mosaicism leads to improved hematology in Fanconi anemia patients.

Fanconi anemia (FA) is characterized by chromosome fragility, bone marrow failure (BMF) and predisposition to cancer. As reverse genetic mosaicism has been described as "natural gene therapy" in patients with FA, we sought to evaluate the clinical course of a cohort of FA mosaic patients followed at referral centers in Spain over a 30-year period. This cohort includes patients with a majority of T cells without chromosomal aberrations in the DEB-chromosomal breakage test. Relative to non-mosaic FA patients, we observed a higher proportion of adult patients in the cohort of mosaics, with a later age of hematologic onset and a milder evolution of (BMF). Consequently, the requirement for hematopoietic stem cell transplant (HSCT) was also lower. Additional studies allowed us to identify a sub-cohort of mosaic FA patients in whom the reversion was present in bone marrow (BM) progenitor cells leading to multilineage mosaicism. These multilineage mosaic patients are older, have a lower percentage of aberrant cells, have more stable hematology and none of them developed leukemia or myelodysplastic syndrome when compared to non-mosaics. In conclusion, our data indicate that reverse mosaicism is a good prognostic factor in FA and is associated with more favorable long-term clinical outcomes.

Optimised molecular genetic diagnostics of Fanconi anaemia by whole exome sequencing and functional studies.

PURPOSE: Patients with Fanconi anaemia (FA), a rare DNA repair genetic disease, exhibit chromosome fragility, bone marrow failure, malformations and cancer susceptibility. FA molecular diagnosis is challenging since FA is caused by point mutations and large deletions in 22 genes following three heritability patterns. To optimise FA patients' characterisation, we developed a simplified but effective methodology based on whole exome sequencing (WES) and functional studies. METHODS: 68 patients with FA were analysed by commercial WES services. Copy number variations were evaluated by sequencing data analysis with RStudio. To test FANCA missense variants, wt FANCA cDNA was cloned and variants were introduced by site-directed mutagenesis. Vectors were then tested for their ability to complement DNA repair defects of a FANCA-KO human cell line generated by TALEN technologies. RESULTS: We identified 93.3% of mutated alleles including large deletions. We determined the pathogenicity of three FANCA missense variants and demonstrated that two FANCA variants reported in mutations databases as 'affecting functions' are SNPs. Deep analysis of sequencing data revealed patients' true mutations, highlighting the importance of functional analysis. In one patient, no pathogenic variant could be identified in any of the 22 known FA genes, and in seven patients, only one deleterious variant could be identified (three patients each with FANCA and FANCD2 and one patient with FANCE mutations) CONCLUSION: WES and proper bioinformatics analysis are sufficient to effectively characterise patients with FA regardless of the rarity of their complementation group, type of mutations, mosaic condition and DNA source.

Screening of BRCA1/2 deep intronic regions by targeted gene sequencing identifies the first germline BRCA1 variant causing pseudoexon activation in a patient with breast/ovarian cancer.

BACKGROUND: Genetic analysis of BRCA1 and BRCA2 for the diagnosis of hereditary breast and ovarian cancer (HBOC) is commonly restricted to coding regions and exon-intron boundaries. Although germline pathogenic variants in these regions explain about ~20% of HBOC cases, there is still an important fraction that remains undiagnosed. We have screened BRCA1/2 deep intronic regions to identify potential spliceogenic variants that could explain part of the missing HBOC susceptibility. METHODS: We analysed BRCA1/2 deep intronic regions by targeted gene sequencing in 192 high-risk HBOC families testing negative for BRCA1/2 during conventional analysis. Rare variants (MAF <0.005) predicted to create/activate splice sites were selected for further characterisation in patient RNA. The splicing outcome was analysed by RT-PCR and Sanger sequencing, and allelic imbalance was also determined when heterozygous exonic loci were present. RESULTS: A novel transcript was detected in BRCA1 c.4185+4105C>T variant carrier. This variant promotes the inclusion of a pseudoexon in mature mRNA, generating an aberrant transcript predicted to encode for a non-functional protein. Quantitative and allele-specific assays determined haploinsufficiency in the variant carrier, supporting a pathogenic effect for this variant. Genotyping of 1030 HBOC cases and 327 controls did not identify additional carriers in Spanish population. CONCLUSION: Screening of BRCA1/2 intronic regions has identified the first BRCA1 deep intronic variant associated with HBOC by pseudoexon activation. Although the frequency of deleterious variants in these regions appears to be low, our study highlights the importance of studying non-coding regions and performing comprehensive RNA assays to complement genetic diagnosis.

Incorporation of semi-quantitative analysis of splicing alterations for the clinical interpretation of variants in BRCA1 and BRCA2 genes.

BRCA1 and BRCA2 (BRCA1/2) genetic variants that disrupt messenger RNA splicing are commonly associated with increased risks of developing breast/ovarian cancer. The majority of splicing studies published to date rely on qualitative methodologies (i.e., Sanger sequencing), but it is necessary to incorporate semi-quantitative or quantitative approaches to accurately interpret the clinical significance of spliceogenic variants. Here, we characterize the splicing impact of 31 BRCA1/2 variants using semi-quantitative capillary electrophoresis of fluorescent amplicons (CE), Sanger sequencing and allele-specific assays. A total of 14 variants were found to disrupt splicing. Allelic-specific assays could be performed for BRCA1 c.302-1G>A and BRCA2 c.516+2T>A, c.1909+1G>A, c.8332-13T>G, c.8332-2A>G, c.8954-2A>T variants, showing a monoallelic contribution to full-length transcript expression that was concordant with semi-quantitative data. The splicing fraction of alternative and aberrant transcripts was also measured by CE, facilitating variant interpretation. Following Evidence-based Network for the Interpretation of Germline Mutant Alleles criteria, we successfully classified eight variants as pathogenic (Class 5), five variants as likely pathogenic (Class 4), and 14 variants as benign (Class 1). We also provide splicing data for four variants classified as uncertain (Class 3), which produced a "leaky" splicing effect or introduced a missense change in the protein sequence, that will require further assessment to determine their clinical significance.

Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification.

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification.

Alternative transcript imbalance underlying breast cancer susceptibility in a family carrying PALB2 c.3201+5G>T.

PURPOSE: Disruption of splicing motifs by genetic variants can affect the correct generation of mature mRNA molecules leading to aberrant transcripts. In some cases, variants may alter the physiological transcription profile composed of several transcripts, and an accurate in vitro evaluation is crucial to establish their pathogenicity. In this study, we have characterized a novel PALB2 variant c.3201+5G>T identified in a breast cancer family. METHODS: Peripheral blood RNA was analyzed in two carriers and ten controls by RT-PCR and Sanger sequencing. The splicing profile was also characterized by semi-quantitative capillary electrophoresis and quantitative PCR. RAD51 foci formation and PALB2 LOH status were evaluated in primary breast tumor samples from the carriers. RESULTS: PALB2 c.3201+5G>T disrupts intron 11 donor splice site and modifies the abundance of several alternative transcripts (∆11, ∆12, and ∆11,12), also present in control samples. All transcripts are predicted to encode for non-functional proteins. Semi-quantitative and quantitative analysis of PALB2 full-length transcript indicated haploinsufficiency in carriers. One tumor exhibited PALB2 LOH and RAD51 assay indicated homologous recombination deficiency in both tumors. CONCLUSIONS: Our results support a pathogenic classification for PALB2 c.3201+5G>T, highlighting the impact of variants causing an imbalanced expression of natural RNA isoforms in cancer susceptibility.

Early Diagnosis of Oral Cancer and Lesions in Fanconi Anemia Patients: A Prospective and Longitudinal Study Using Saliva and Plasma.

Fanconi anemia (FA) patients display an exacerbated risk of oral squamous cell carcinoma (OSCC) and oral potentially malignant lesions (OPMLs) at early ages. As patients have defects in their DNA repair mechanisms, standard-of-care treatments for OSCC such as radiotherapy and chemotherapy, give rise to severe toxicities. New methods for early diagnosis are urgently needed to allow for treatment in early disease stages and achieve better clinical outcomes. We conducted a prospective, longitudinal study wherein liquid biopsies from sixteen patients with no clinical diagnoses of OPML and/or OSCC were analyzed for the presence of mutations in cancer genes. The DNA from saliva and plasma were sequentially collected and deep-sequenced, and the clinical evaluation followed over a median time of approximately 2 years. In 9/16 FA patients, we detected mutations in cancer genes (mainly TP53) with minor allele frequencies (MAF) of down to 0.07%. Importantly, all patients that had mutations and clinical follow-up data after mutation detection (n = 6) developed oral precursor lesions or OSCC. The lead-time between mutation detection and tumor diagnosis ranged from 23 to 630 days. Strikingly, FA patients without mutations displayed a significantly lower risk of developing precursor lesions or OSCCs. Therefore, our diagnostic approach could help to stratify FA patients into risk groups, which would allow for closer surveillance for OSCCs or precursor lesions.

An spanish study of secondary findings in families affected with mendelian disorders: choices, prevalence and family history.

Clinical exome sequencing has the potential to identify pathogenic variants unrelated to the purpose of the study (secondary findings, SFs). Data describing actual choices of SFs in participants in a clinical setting and factors influencing their decision are virtually non-existant in Europe. In this work, we report the acceptance rate of SFs, calculate their prevalence and study factors associated with the decision in a cohort of patients affected with a rare genetic disorder in a Spanish Hospital. Finally, we re-examine the presence of previously non reported family history in positive cases. We retrospectively reviewed informed consent choices and SF results from 824 unrelated probands affected with rare genetic disorders who underwent whole-genome or exome sequencing. Ninety percent of families (740/824) affected with rare disorders wished to be informed of SFs. Declining SFs was associated with a prenatal setting (30% vs. 8.7%, p = 0.025), consanguinity (19% vs. 8.7%, p = 0.013), male gender (10.6% vs. 1.5%, p = 0.00865) and the proband being a minor (10.6% vs. 1.5%, p = 0.014). Overall, 27 pathogenic or likely pathogenic variants were identified in 27 individuals, with an SF prevalence of 3.6%. Disclosure of SFs increased the percentage of positive family histories and resulted in early diagnosis or changes in the management of 10 individuals from five families. We show that the acceptance of SFs in Spain is high and the disclosure of SFs leads to a clinically meaningful change in the medical management of individuals.

Early-Stage Breast Cancer Detection in Breast Milk.

Breast cancer occurring during pregnancy (PrBC) and postpartum (PPBC) is usually diagnosed at more advanced stages compared with other breast cancer, worsening its prognosis. PPBC is particularly aggressive, with increased metastatic risk and mortality. Thus, effective screening methods to detect early PrBC and PPBC are needed. We report for the first time that cell-free tumor DNA (ctDNA) is present in breast milk (BM) collected from patients with breast cancer. Analysis of ctDNA from BM detects tumor variants in 87% of the cases by droplet digital PCR, while variants remain undetected in 92% of matched plasma samples. Retrospective next-generation sequencing analysis in BM ctDNA recapitulates tumor variants, with an overall clinical sensitivity of 71.4% and specificity of 100%. In two cases, ctDNA was detectable in BM collected 18 and 6 months prior to standard diagnosis. Our results open up the potential use of BM as a new source for liquid biopsy for PPBC detection. SIGNIFICANCE: For the first time, we show that BM obtained from patients with breast cancer carries ctDNA, surpassing plasma-based liquid biopsy for detection and molecular profiling of early-stage breast cancer, even prior to diagnosis by image. See related commentary by Cunningham and Turner, p. 2125. This article is featured in Selected Articles from This Issue, p. 2109.

Anogenital distance and the risk of prostate cancer.

UNLABELLED: Study Type - Prognosis (cohort) Level of Evidence 2b What's known on the subject? and What does the study add? In animals, anogenital distance has been shown to be related to the action of fetal androgens, and exposure to chemicals such as dioxins that exhibit antiandrogenic activity results in shorter distances in male rats. In studies conducted in children, anogenital distance has been associated with endocrine disruptors such as phthalates. Studies conducted in young adults found that a shorter anoscrotal distance was a predictor of low sperm concentration, and a longer anoscrotal distance was associated with fatherhood, a higher sperm density and a higher total motile sperm count. The present study is the first to report anogenital measurements in adults in relation to the risk of cancer, showing that a phenotype reflecting normal in utero sexual development in males is associated with a lower risk of prostate cancer. There are two published studies evaluating sperm quality and fatherhood suggesting a connecting mechanism related to the disruption of androgen-mediated pathways in utero that affects reproductive potential and the risk of prostate cancer. OBJECTIVES: • To measure anogenital distance in patients with prostate cancer and control subjects without cancer. • To evaluate the association of anogenital distance with prostate cancer in a case-control study in Spain. MATERIALS AND METHODS: • Anogenital distances from anus to upper penis (AGDAP ) and from anus to scrotum (AGDAS ) were measured in 60 patients with prostate cancer in two hospitals in Barcelona and in 52 urological controls. • Each measurement was performed three times by the same trained examiner using a digital caliper RESULTS: • Patients had an ≈5 mm shorter AGDAP than controls, whereas no difference was observed for AGDAS . • A higher AGDAP was associated with a lower risk of prostate cancer, with an adjusted odds ratio per 5 mm increase in AGDAP of 0.83 (95% confidence interval, 0.70-0.99, P= 0.03). CONCLUSIONS: • The present study is the first to report anogenital measurements in adults in relation to the risk of cancer. • The present study showed that a phenotype reflecting normal in utero sexual development in men is associated with a lower risk of prostate cancer. • There are two published studies (Mendiola et al. Environ Health Perspect 2011; 119: 958-63; Eisenberg et al. PLoS One 2011; 6: e18973) evaluating sperm quality and fatherhood suggesting a connecting mechanism related to the disruption of androgen-mediated pathways in utero that affects reproductive potential and the risk of prostate cancer.

Development of a mouse model for spontaneous oral squamous cell carcinoma in Fanconi anemia.

Fanconi anemia (FA) patients frequently develop oral squamous cell carcinoma (OSCC). This cancer in FA patients is diagnosed within the first 3-4 decades of life, very often preceded by lesions that suffer a malignant transformation. In addition, they respond poorly to current treatments due to toxicity or multiple recurrences. Translational research on new chemopreventive agents and therapeutic strategies has been unsuccessful partly due to scarcity of disease models or failure to fully reproduce the disease. Here we report that Fanca gene knockout mice (Fanca-/-) frequently display pre-malignant lesions in the oral cavity. Moreover, when these animals were crossed with animals having conditional deletion of Trp53 gene in oral mucosa (K14cre;Trp53F2-10/F2-10), they spontaneously developed OSCC with high penetrance and a median latency of less than ten months. Tumors were well differentiated and expressed markers of squamous differentiation, such as keratins K5 and K10. In conclusion, Fanca and Trp53 genes cooperate to suppress oral cancer in mice, and Fanca-/-;K14cre;Trp53F2-10/F2-10 mice constitute the first animal model of spontaneous OSCC in FA.

BRCA1 and BRCA2 whole cDNA analysis in unsolved hereditary breast/ovarian cancer patients.

Germline pathogenic variants in BRCA1 and BRCA2 genes (BRCA1/2) explain an important fraction of hereditary breast/ovarian cancer (HBOC) cases. Genetic testing generally involves examining coding regions and exon/intron boundaries, thus the frequency of deleterious variants in non-coding regions is unknown. Here we analysed BRCA1/2 whole cDNA in a large cohort of 320 unsolved high-risk HBOC cases in order to identify potential splicing alterations explained by variants in BRCA1/2 deep intronic regions. Whole RNA splicing profiles were analysed by RT-PCR using Sanger sequencing or high-resolution electrophoresis in a QIAxcel instrument. Known predominant BRCA1/2 alternative splicing events were detected, together with two novel events BRCA1 ▼21 and BRCA2 Δ18q_27p. BRCA2 exon 3 skipping was detected in one patient (male) affected with breast cancer, caused by a known Portuguese founder mutation (c.156_157insAluYa5). An altered BRCA2 splicing pattern was detected in three patients, consisting in the up-regulation of ▼20A, Δ22 and ▼20A+Δ22 transcripts. In silico analysis and semi-quantitative data identified the polymorphism BRCA2 c.8755-66T>C as a potential modifier of Δ22 levels. Our findings suggest that mRNA alterations in BRCA1/2 caused by deep intronic variants are rare in Spanish population. However, RNA analysis complements DNA-based strategies allowing the identification of alterations that could go undetected by conventional testing.

Clinical consequences of BRCA2 hypomorphism.

The tumor suppressor FANCD1/BRCA2 is crucial for DNA homologous recombination repair (HRR). BRCA2 biallelic pathogenic variants result in a severe form of Fanconi anemia (FA) syndrome, whereas monoallelic pathogenic variants cause mainly hereditary breast and ovarian cancer predisposition. For decades, the co-occurrence in trans with a clearly pathogenic variant led to assume that the other allele was benign. However, here we show a patient with biallelic BRCA2 (c.1813dup and c.7796 A > G) diagnosed at age 33 with FA after a hypertoxic reaction to chemotherapy during breast cancer treatment. After DNA damage, patient cells displayed intermediate chromosome fragility, reduced survival, cell cycle defects, and significantly decreased RAD51 foci formation. With a newly developed cell-based flow cytometric assay, we measured single BRCA2 allele contributions to HRR, and found that expression of the missense allele in a BRCA2 KO cellular background partially recovered HRR activity. Our data suggest that a hypomorphic BRCA2 allele retaining 37-54% of normal HRR function can prevent FA clinical phenotype, but not the early onset of breast cancer and severe hypersensitivity to chemotherapy.

Paired Somatic-Germline Testing of 15 Polyposis and Colorectal Cancer-Predisposing Genes Highlights the Role of APC Mosaicism in de Novo Familial Adenomatous Polyposis.

Familial adenomatous polyposis (FAP) is an autosomal dominant syndrome responsible for 1% of colorectal cancers (CRCs). Up to 90% of classic FAPs are caused by inactivating mutations in APC, and mosaicism has been previously reported in 20% of de novo cases, usually linked to milder phenotypic manifestations. This study aimed to explore the prevalence of mosaicism in 11 unsolved cases of classic FAP and to evaluate the diagnostic yield of somatic testing. Paired samples of colorectal polyps, tumors, and/or mucosa were analyzed using a custom next-generation sequencing panel targeting 15 polyposis and CRC-predisposing genes. Whenever possible, the extension of mosaicism to blood or sperm was also examined. Of 11 patients with classic adenomatous polyposis, a mosaic pathogenic variant in APC was identified in 7 (64%). No other altered genes were identified. In two of seven patients (29%), mosaicism was found restricted to colonic tissues, whereas in five of seven patients (71%), it was extended to the blood. Germline affectation was confirmed in one patient. We report the first analysis at a somatic level of 15 genes associated with CRC susceptibility, which highlights the role of APC mosaicism in classic FAP etiology. The results further reinforce the importance of testing target tissues when blood test results are negative.