miRNA deregulation and relationship with metabolic parameters after Mediterranean dietary intervention in BRCA-mutated women.

Background: Breast cancer onset is determined by a genetics-environment interaction. BRCA1/2 gene alterations are often genetically shared in familial context, but also food intake and hormonal assessment seem to influence the lifetime risk of developing this neoplasia. We previously showed the relationship between a six-months Mediterranean dietary intervention and insulin, glucose and estradiol levels in BRCA1/2 carrier subjects. The aim of the present study was to evidence the eventual influence of this dietary intervention on the relationship between circulating miRNA expression and metabolic parameters in presence of BRCA1/2 loss of function variants. Methods: Plasma samples of BRCA-women have been collected at the baseline and at the end of the dietary intervention. Moreover, subjects have been randomized in two groups: dietary intervention and placebo. miRNA profiling and subsequent ddPCR validation have been performed in all the subjects at both time points. Results: ddPCR analysis confirmed that five (miR-185-5p, miR-498, miR-3910, miR-4423 and miR-4445) of seven miRNAs, deregulated in the training cohort, were significantly up-regulated in subjects after dietary intervention compared with the baseline measurement. Interestingly, when we focused on variation of miRNA levels in the two timepoints, it could be observed that miR-4423, miR-4445 and miR-3910 expressions are positively correlated with variation in vitaminD level; whilst miR-185-5p difference in expression is related to HDL cholesterol variation. Conclusions: We highlighted the synergistic effect of a healthy lifestyle and epigenetic regulation in BC through the modulation of specific miRNAs. Different miRNAs have been reported involved in the tumor onset acting as tumor suppressors by targeting tumor-associated genes that are often downregulated.

Randomized Placebo Controlled Trial of Low-Dose Tamoxifen to Prevent Recurrence in Breast Noninvasive Neoplasia: A 10-Year Follow-Up of TAM-01 Study.

PURPOSE: Five-year data of the phase III trial TAM-01 showed that low-dose tamoxifen at 5 mg once daily administered for 3 years in women with intraepithelial neoplasia (IEN) reduced by 52% the recurrence of invasive breast cancer or ductal carcinoma in situ (DCIS), without additional adverse events over placebo. Here, we present the 10-year results. METHODS: We randomly assigned 500 women with breast IEN (atypical ductal hyperplasia, lobular carcinoma in situ [LCIS], or hormone-sensitive or unknown DCIS) to low-dose tamoxifen or placebo after surgery with or without irradiation. The primary end point was the incidence of invasive breast cancer or DCIS. RESULTS: The TAM-01 population included 500 women (20% atypical ductal hyperplasia, 11% LCIS, and 69% DCIS). The mean (±SD) age at the start of treatment was 54 ± 9 years, and 58% of participants were postmenopausal. After a median follow-up of 9.7 years (IQR, 8.3-10.9 years), 66 breast cancers (15 in situ; 51 invasive) were diagnosed: 25 in the tamoxifen group and 41 in the placebo group (annual rate per 1,000 person-years, 11.3 with tamoxifen v 19.5 with placebo; hazard ratio [HR], 0.58; 95% CI, 0.35 to 0.95; log-rank P = .03). Most recurrences were invasive (77%) and ipsilateral (59%). Regarding contralateral breast cancer incidence, there were six events in the tamoxifen arm and 16 in the placebo arm (HR, 0.36; 95% CI, 0.14 to 0.92; P = .025). The number needed to be treated to prevent one case of breast event with tamoxifen therapy was 22 in 5 years and 14 in 10 years. The benefit was seen across all patient subgroups. There was a significant 50% reduction of recurrence with tamoxifen in the DCIS cohort, which represents 70% of the overall population (HR, 0.50; 95% CI, 0.28 to 0.91; P = .02). No between-group difference in the incidence of serious adverse events was reported during the prolonged follow-up period. CONCLUSION: Tamoxifen 5 mg once daily for 3 years significantly prevents recurrence from noninvasive breast cancer after 7 years from treatment cessation without long-term adverse events.

Remote vs in-person BRCA1/2 non-carriers test disclosure: patients' choice during Covid-19 pandemic restriction.

During Covid-19 pandemic most hospitals have restricted in-person delivery of non-essential healthcare services, including genetic testing delivery, to slow the spread of the virus. Our Onco-Genetic Service also faced this challenging period and had to re-organize its clinical practice with the use of tele-health. Aim of the present paper is to understand whether and how Covid-19-related changes in medical practice influenced patients' satisfaction about the health service provided. 125 BRCA1/2 non carriers (109/125, 87.2% female and 16/125, 12.8% male) in Istituto Tumori "Giovanni Paolo II" of Bari were enrolled. All participants were asked to choose whether they prefer in-person or remote post-test counselling session. Basing on patients' choice, two groups of subjects were composed. One week after the post-test counselling session, participants were phone called and asked to complete: a socio-demographic form, a brief structured interview about their Covid-19 related worries and their satisfaction with the health service provided, Hospital Anxiety and Depression Scale and Fear of Covid-19 scale. Qualitative information about patients' choice were also collected. No significant difference about patients' satisfaction with the health service provided emerged between groups. Patients who preferred remote post-test counselling had higher anxiety, worries and fear-of Covid-19 than the others. All remote-counselling subjects preferred tele-genetics because of Covid-19 security, would choose it again and would recommend it to others. Cancer tele-genetics offers good guarantees of comfort and efficacy, but patients' choices are related to personal and psychological variables. The use of tele-genetics has to be a patient's choice.

Spectrum of Germline Pathogenic Variants in BRCA1/2 Genes in the Apulian Southern Italy Population: Geographic Distribution and Evidence for Targeted Genetic Testing.

BRCA1/2-associated hereditary breast and ovarian cancer is the most common form of hereditary breast and ovarian cancer and occurs in all ethnicities and racial populations. Different BRCA1/BRCA2 pathogenic variants (PVs) have been reported with a wide variety among populations. In this study, we retrospectively analyzed prevalence and geographic distribution of pathogenic germline BRCA1/2 variants in families from Apulia in southern Italy and evaluated the genotype-phenotype correlations. Data were collected from Oncogenetic Services present in Apulian hospitals and a shared database was built containing Apulian native probands (n = 2026) that had undergone genetic testing from 2004 to 2019. PVs were detected in 499 of 2026 (24.6%) probands and 68.5% of them (342 of 499) were in the BRCA1 gene. We found 65 different PVs in BRCA1 and 46 in BRCA2. There were 10 most recurrent PVs and their geographical distribution appears to be significantly specific for each province. We have assumed that these PVs are related to the historical and geopolitical changes that occurred in Apulia over time and/or to a "founder effect". Broader knowledge of BRCA1/2 prevalence and recurring PVs in specific geographic areas could help establish more flexible genetic testing strategies that may enhance our ability to detect high-risk subjects.

Effect Modifiers of Low-Dose Tamoxifen in a Randomized Trial in Breast Noninvasive Disease.

PURPOSE: Low-dose tamoxifen halved recurrence after surgery in a phase III trial in breast noninvasive disease without increasing adverse events. We explored the effect of low-dose tamoxifen in clinically relevant subgroups, including menopausal status, estradiol levels, smoking, body mass index, and proliferation of baseline lesion. PATIENTS AND METHODS: Incidence of invasive breast cancer or ductal carcinoma in situ was the primary endpoint. HRs and interaction terms were estimated using Cox models. RESULTS: A favorable HR and 95% confidence interval (CI) could be demonstrated for postmenopausal status (HR = 0.30; 95% CI, 0.11-0.82 vs. HR = 0.73; 95% CI, 0.30-1.76 in premenopausal women; P interaction = 0.13), women with estradiol less than 15.8 pg/mL, presence of menopausal symptoms at baseline, and never smoking (P interaction = 0.07), although the interaction P value was >0.05 for all characteristics. Efficacy was similar in all body mass index categories. Tumors with Ki-67 above the median level of 10% had a greater benefit (HR = 0.27; 95% CI, 0.09-0.81) than those with Ki-67 ≤10% (HR = 1.58; 95% CI, 0.45-5.60; P interaction = 0.04). CONCLUSIONS: The efficacy of low-dose tamoxifen seems to be greater in postmenopausal women and in women with lower estradiol levels. Benefits appear to be larger also in women with menopausal symptoms, never smokers, and tumors with Ki-67 >10%. Our results by menopausal status provide important insight into low-dose tamoxifen personalized treatment, although caution is necessary given their exploratory nature. Observation of an improved response in tumors with Ki-67 >10% is consistent but the use of the marker in this setting is investigational.See related commentary by Fabian, p. 3510.

Lifestyle Characteristics in Women Carriers of BRCA Mutations: Results From an Italian Trial Cohort.

BACKGROUND: Women with deleterious mutations in BRCA1/2 have a high lifetime penetrance of developing breast cancer and/or ovarian cancer. Genetic and/or environmental factors may influence BRCA penetrance, and identifying modifiable exposures might be valuable for prevention. PATIENTS AND METHODS: We implemented a multicenter prospective 2-arm (1:1) randomized controlled trial to investigate whether a Mediterranean dietary intervention with moderate protein restriction would reduce potential modulators of BRCA penetrance such as insulin-like growth factor 1 (IGF-1), body weight, and metabolic risk factors. We studied the baseline characteristics of women with BRCA-positive disease who joined the trial cohort, focusing on the relationships between selected lifestyle exposures, metabolic/anthropometric parameters, and BRCA-related cancer. RESULTS: A total of 502 women (304 with a previous diagnosis of breast cancer and/or ovarian cancer and 198 unaffected) with deleterious BRCA mutations, with or without a previous cancer, aged 18 to 70 years and without metastases were included. Late age at menarche and pregnancy were negatively associated with BRCA-related cancer, especially in women with BRCA1-positive disease. Higher fat mass and the presence of 4 or 5 metabolic risk factors were significantly associated with BRCA-related cancer (hazard ratio, 1.87, 95% confidence interval, 1.21-2.88; and hazard ratio, 1.87, 95% confidence interval, 1.11-3.19, respectively), with greater effect in BRCA2-positive women. CONCLUSIONS: Our findings confirm previous observations about reproductive factors in women with BRCA disease and suggest a potential impact of metabolic factors in BRCA-related cancer. The prospective follow-up of the trial cohort will enable us to study the environmental modulators of BRCA penetrance and their impact in relation to the history of BRCA-related cancer. [ClinicalTrials.gov NCT03066856].

Somatic BRCA Mutation in a Cholangiocarcinoma Patient for HBOC Syndrome Detection.

BRCA-associated hereditary breast and ovarian cancer syndrome (HBOC) is characterized by an increased risk of developing other malignancies including cholangiocarcinoma (CCA). Somatic BRCA mutations have been reported in CCA, but they have yet to be utilized in a proband case to identify HBOC in families. Two healthy daughters of a deceased female patient who had had metachronous breast cancer and CCA received genetic counseling to assess their cancer risk. Somatic BRCA1/2 mutation analysis was performed by next-generation sequencing on the DNA extracted from a formalin-fixed, paraffin-embedded CCA biopsy specimen of their mother. A pathogenic variant was identified (c.6468_6469delTC in a BRCA2 gene mutation). Germline BRCA mutation analysis of the two daughters detected the same pathogenic variant in one of them. For the first time, a CCA somatic BRCA mutation has been used to identify a family with HBOC.

The Role of Circulating Adiponectin and SNP276G>T at ADIPOQ Gene in BRCA-mutant Women.

BACKGROUND: Environmental factors may influence the lifetime risk of cancer (penetrance) in women with a BRCA mutation. MATERIALS AND METHODS: In 89 BRCA-mutant women, affected or unaffected by breast/ovarian cancer, we explored serum levels of adipokines and their relation with the polymorphism SNP276G>T as modulators of BRCA penetrance. RESULTS: Affected women had significantly lower adiponectin than healthy women. Affected women with rs1501299 TT had significantly lower adiponectin and higher leptin than GT and GG genotypes. GT genotype was significantly associated with the disease status [odds ratio (OR)=3.24, 95% confidence interval (95% CI)=1.03-10.17]. Women in the lower tertile of serum adiponectin had a RR of BRCA-associated cancer of 2.80, 95% CI=1.1-7.1 (p for trend=0.03) compared with women in the higher tertile. CONCLUSION: In the SNP rs1501299 the T allele was significantly associated with lower serum levels of adiponectin in affected women, suggesting that the T allele might be related to cancer.

Oncological genetic counseling (OGC) for high-risk hereditary cancer: what can hospital anxiety and depression scale (HADs) tell us?

PURPOSE: This study aimed to verify whether and how anxiety and depression symptoms are associated both to socio-demographic and clinical variables (age, civil status, type of cancer diagnosed, time elapsed between cancer diagnosis and Oncologic Genetic Counseling/OGC, number of relatives affected by cancer) and to psychological features (presence/absence of previous psychological suffering), subjective cancer risk perception, psychological attitude approaching/OGC) in a sample of Caucasian patients accessing OGC. METHODS: 201 participants (193 female and 8 male) accessing OGC in the Istituto di Ricovero e Cura Carattere Scientifico (IRCCS) Giovanni Paolo II in Bari completed the Hospital Depression and Anxiety Scale (HADs) that was analyzed as global scoring, anxiety (HAD-A) and depression subscale (HAD-D). RESULTS: In our sample, higher HADs, HAD-A and HAD-D scorings were associated in different ways to both socio-demographic information (age: p value 0.019), clinical and medical features (personal history of cancer: HAD-D p value 0.02; months elapsed between diagnosis and OGC, HAD-A p value 0.004 and HADs p value 0.008) and psychological dimensions (approaching genetic counseling: anxiety p value 0.06; fear p value 0.02; duty p value 0.04). CONCLUSION: This study showed that during the process of oncological genetic counseling the importance of taking into consideration not only medical variables but also cognitive and emotional aspects from both the individual and family spheres, in order to assure adequate care of the patient.

Randomized Placebo Controlled Trial of Low-Dose Tamoxifen to Prevent Local and Contralateral Recurrence in Breast Intraepithelial Neoplasia.

PURPOSE: Tamoxifen administered for 5 years at 20 mg/d is effective in breast cancer treatment and prevention, but toxicity has limited its broad use. Biomarker trials showed that 5 mg/d is not inferior to 20 mg/d in decreasing breast cancer proliferation. We hypothesized that a lower dose given for a shorter period could be as effective in preventing recurrence from breast intraepithelial neoplasia but have a lower toxicity than the standard dose. PATIENTS AND METHODS: We conducted a multicenter randomized trial of tamoxifen, 5 mg/d or placebo administered for 3 years after surgery in women with hormone-sensitive or unknown breast intraepithelial neoplasia, including atypical ductal hyperplasia and lobular or ductal carcinoma in situ. The primary end point was the incidence of invasive breast cancer or ductal carcinoma in situ. RESULTS: Five hundred women 75 years of age or younger were included. After a median follow-up of 5.1 years (interquartile range, 3.9-6.3 years), there were 14 neoplastic events with tamoxifen and 28 with placebo (11.6 v 23.9 per 1,000 person-years; hazard ratio, 0.48; 95% CI, 0.26 to 0.92; P = .02), which resulted in a 5-year number needed to treat of 22 (95% CI, 20 to 27). Tamoxifen decreased contralateral breast events by 75% (three v 12 events; hazard ratio, 0.25; 95% CI, 0.07 to 0.88; P = .02). Patient-reported outcomes were not different between arms except for a slight increase in frequency of daily hot flashes with tamoxifen (P = .02). There were 12 serious adverse events with tamoxifen and 16 with placebo, including one deep vein thrombosis and one stage I endometrial cancer with tamoxifen and one pulmonary embolism with placebo. CONCLUSION: Tamoxifen at 5 mg/d for 3 years can halve the recurrence of breast intraepithelial neoplasia with a limited toxicity, which provides a new treatment option in these disorders.

Adipokines in hereditary breast cancer patients and healthy relatives.

Background: The role of adipocytokines and ghrelin in hereditary breast cancer syndrome (HBCS) has never been tested. Results: No significant differences in leptin, adiponectin and ghrelin plasma levels between cancer patients and healthy subjects was observed. Conversely, an higher level of adiponectin was shown in healthy subjects with BRCA 1/2 gene mutation vs those without (p < 0.03). Logistic regression analysis demonstrated that Adiponectin plasma level (OR 0.26; 95% CI:0.007-0.81; p < 0.02) and age (OR 5.51; 95% CI:1.78-19.71; p < 0.004) were the only factors independently associated with BMI; furthermore, Leptin plasma level (OR 0.23; 95% CI:0.06-0.76; p < 0.01) and age (OR 0.05; 95% CI:0.05-0.61; p < 0.007) resulted the only factors significantly associated with breast cancer. Materials and Methods: We analyzed blood plasma expression of leptin, adiponectin and ghrelin using Bio-Plex platform in 25 breast cancer patients with HBCS and in 38 healthy relatives. BRCA 1/2 gene status (presence of pathogenic mutations by direct molecular sequencing), clinical-pathological characteristics and Body Mass Index (BMI) of each subject were recorded. Conclusions: Adiponectin confirms to be associated with BMI also in subjects with HBCS. Leptin plasma level seems a direct and independent biomarker of a breast cancer risk. A validation of Leptin as a circulating biomarker of breast cancer development in larger series of HBCS subjects is needed.

TGFbeta and miRNA regulation in familial and sporadic breast cancer.

The term 'BRCAness' was introduced to identify sporadic malignant tumors sharing characteristics similar to those germline BRCA-related. Among all mechanisms attributable to BRCA1 expression silencing, a major role has been assigned to microRNAs. MicroRNAs role in familial and sporadic breast cancer has been explored but few data are available about microRNAs involvement in homologous recombination repair control in these breast cancer subgroups. Our aim was to seek microRNAs associated to pathways underlying DNA repair dysfunction in breast cancer according to a family history of the disease. Affymetrix GeneChip microRNA Arrays were used to perform microRNA expression analysis in familial and sporadic breast cancer. Pathway enrichment analysis and microRNA target prediction was carried out using DIANA miRPath v.3 web-based computational tool and miRWalk v.2 database. We analyzed an external gene expression dataset (E-GEOD-49481), including both familial and sporadic breast cancers. For microRNA validation, an independent set of 19 familial and 10 sporadic breast cancers was used. Microarray analysis identified a signature of 28 deregulated miRNAs. For our validation analyses by real time PCR, we focused on miR-92a-1*, miR-1184 and miR-943 because associated to TGF-ß signalling pathway, ATM and BRCA1 genes expression. Our results highlighted alterations in miR-92a-1*, miR-1184 and miR-943 expression levels suggesting their involvement in repair of DNA double-strand breaks through TGF-beta pathway control.

BRCA1/2 and clinical outcome in a monoinstitutional cohort of women with hereditary breast cancer.

The clinical outcome of BRCA mutation carriers and non-carriers still remains a topic of discussion. In order to interpret controversial data, in the present study, we analyzed a large consecutive monoinstitutional series of breast cancer patients and relatives with familial features carrying or not carrying BRCA mutations. The intense research in recent years regarding the clinical genetics of patients with breast or ovarian cancer and their relatives has allowed the organization of a unique database comprising anamnestic, clinical, pathological and molecular data. Families with two or more cases of breast cancer under the age of 50 years, or with three cases at any age, were identified. From June, 2003 to June, 2010, a total of 202 patients (136 probands + 66 relatives) from 45 families were included in the analysis. A total of 136 (49 carrier and 87 non-carrier) cases had a cancer diagnosis at the time of their genetic testing. Twenty and 24 events were observed in the carrier and control group, respectively. The 10-year disease-free suvival rate was 57% for patients in the control group compared with 50% for patients carrying a BRCA mutation (P=0.15 by log-rank test). Finally, 66 (32 genetic and 34 control) cases were unaffected at the time of molecular analysis, and 6 new cases of cancer were observed in the carriers, while no new cases were detected in the control cohort. Thus, at age 50, 40% of carriers had a high risk of disease (P=0.0069 by log-rank test). Our data support the hypothesis that the presence of BRCA mutations does not alter the clinical outcome for hereditary breast cancer patients. Conversely, BRCA mutations are proven to be crucial for prediction of risk in healthy relatives from carrier families.

Molecular and in silico analysis of BRCA1 and BRCA2 variants.

Germline mutations of high penetrant BRCA1 and BRCA2 genes have been associated to hereditary breast cancer risk, while polymorphic variants of the two genes still have an unknown role in breast pathogenesis. The aim of our study was to characterize BRCA1 and BRCA2 genes polymorphic variants in familial breast cancer. 110 patients affected by familial breast and/or ovarian cancer have been consecutively enrolled according to family history and BRCA mutation risk. All of them have been screened for BRCA1 and BRCA2 pathogenetic mutations, SNPs and intronic variants. In silico analysis have been also performed using different computational methods to individualize genetic variations that can alter the two genes expression and function. BRCA1 resulted mutated in 14% while BRCA2 in 3% of cases, while 80% of patients presented at least one polymorphism. A neural network splicing prediction model individualized one BRCA1 and one BRCA2 intronic variants able to determine alternative splicing. Furthermore, Q356R BRCA1 and N289H BRCA2 appear to show a possible harmful role also due to their location in functional regions of the two genes. However, in silico data are not always consistent with biological evidences. In conclusion, SNPs profile provides a basis for DNA-based cancer risk classification and help to define the gene alterations that could influence biochemistry activity protein or could modify drug sensitivity.

Analysis of the reasons for accepting or declining participation in genetic research for breast cancer: a hospital-based population study.

The majority of a hospital-based population accepted to participate in a molecular screening project for familial breast cancer, giving their informed consent to blood sampling. Only 9.5% of patients declined to sign the consent form. Here we report the reasons for refusal and we critically review our methodological approach to obtain consent for a blood test for genetic research in a clinical setting.