Bernardo Bonanni’s research while affiliated with IEO - Istituto Europeo di Oncologia and other places

Lynch syndrome is a genetic condition predisposing to cancer, particularly colorectal cancer and endometrial cancer, due to germline mutations in MisMatch Repair genes. More rarely, Lynch syndrome is the result of a constitutional MLH1 promoter methylation. This review summarizes the current knowledge about the role of this epigenetic mechanism in the Lynch syndrome. Universal Tumor Screening, performed on tumoral specimens to identify features suggestive of Lynch syndrome, shows the same features both in the case of sporadic cancers and Lynch syndrome-cancers due to a constitutional MLH1 methylation: microsatellite instability, deficiency of MisMatch Repair proteins, and methylation of MLH1 gene. Over the last few years, identifying methylation of MLH1 promoter on tumors was used to discern sporadic tumors from Lynch syndrome tumors: the methylation of the MLH1 promoter was usually explained as a somatic event and this could lead to a missed diagnosis of some Lynch syndrome cases. Therefore, establishing criteria to decide when to test patients for constitutional MLH1 methylation is urgent. In the case of microsatellite instability/deficiency of MisMatch Repair tumors with MLH1 methylation, a germline genetic test could be requested for all colorectal cancer patients aged 55 years or younger and all endometrial cancer patients younger than 50 years old, independently from family history. The prevalence of germline MLH1 epimutations is not precisely known and possibly underestimated. The associated cancer risk could be similar to that due to a MLH1 sequence variant. MLH1 epimutations could be secondary to other genetic defects and follow an autosomal dominant inheritance. On the contrary, primary epimutations are often “ de novo” events, and their transmission does not follow Mendelian rules.

Women carrying pathogenic/likely pathogenic (P/LP) variants in moderate- or high-penetrance genes have an increased risk of developing breast cancer. However, most P/LP variants associated with breast cancer risk show incomplete penetrance. Age, gender, family history, polygenic risk, lifestyle, reproductive, hormonal, and environmental factors can affect the expressivity and penetrance of the disease. However, there are gaps in translating how individual genomic variation affects phenotypic presentation. The expansion of criteria for genetic testing and the increasing utilization of comprehensive genetic panels may enhance the identification of individuals carrying P/LP variants linked to hereditary breast cancer. Individualized risk assessment could facilitate the implementation of personalized risk-reduction strategies for these individuals. Preventive interventions encompass lifestyle modifications, chemoprevention, enhanced surveillance through breast imaging, and risk-reducing surgeries. This review addresses the current literature’s inconsistencies and limitations, particularly regarding risk factors and the intensity of preventive strategies for women with P/LP variants in moderate- and high-penetrance genes. In addition, it synthesizes the latest evidence on risk assessment and primary and secondary prevention in women at high risk of breast cancer.

Women carrying pathogenic/likely pathogenic (P/LP) variants in moderate- or high-penetrance genes have an increased risk of developing breast cancer. However, most P/LP variants associated with breast cancer risk show incomplete penetrance. Age, gender, family history, polygenic risk, lifestyle, reproductive, hormonal, and environmental factors can affect the expressivity and penetrance of the disease. However, there are gaps in translating how individual genomic variation affects phenotypic presentation. The expansion of criteria for genetic testing and the increasing utilization of comprehensive genetic panels may enhance the identification of individuals carrying P/LP variants linked to hereditary breast cancer. Individualized risk assessment could facilitate the implementation of personalized risk-reduction strategies for these individuals. Preventive interventions encompass lifestyle modifications, chemoprevention, enhanced surveillance through breast imaging, and risk-reducing surgeries. This review addresses the current literature's inconsistencies and limitations, particularly regarding risk factors and the intensity of preventive strategies for women with P/LP variants in moderate- and high-penetrance genes. In addition, it synthesizes the latest evidence on risk assessment and primary and secondary prevention in women at high risk of breast cancer.

Background Breast Cancer (BC) prevention strategies range from lifestyle changes such as increasing physical activity and reducing body weight to preventive drugs like tamoxifen, known to reduce BC incidence in high-risk women. Sex Hormone Binding Globulin (SHBG) is related to BC risk due to its ability to bind circulating estradiol at high affinity and to regulate estradiol action. A study protocol is presented based on the assessment of the effect of different interventions such as tamoxifen at 10 mg every other day (LDT), intermittent caloric restriction (ICR) two days per week, lifestyle intervention (LI, step counter use) and their combination on the modulation of SHBG and several other biomarkers associated to BC. Methods A randomized phase II biomarker study will be conducted in 4 Italian centers. Unaffected women aged between 18 and 70 years, carriers of a germline pathogenetic variant (BRCA1, BRCA2, PALB2, or other moderate penetrance genes), or with a >5% BC risk at 10 years (according to the Tyrer-Cuzick or the Breast Cancer Surveillance Consortium Risk models) or with a previous diagnosis of intraepithelial neoplasia will be eligible. A total of 200 participants will be randomized to one of the four arms: LDT; LDT + ICR; LI; LI + ICR. Interventions will span six months, with baseline and follow-up clinic visits and interim phone calls. Discussion The aim of the study is to verify whether LDT increases circulating SHBG more than LI with or without ICR after 6 months. Secondary objectives include assessing HOMA-index, inflammatory markers, adiponectin/leptin ratio, quality of life (QoL), safety, toxicity, mammographic density, and changes in microbiome composition across groups. The study’s innovation lies in its inclusion of diverse BC risk categories and combination of pharmaceutical and behavioral interventions, potentially enhancing intervention efficacy while balancing tamoxifen’s side effects on QoL, especially menopausal symptoms. Trial registration EuCT number:2023-503994-39-00; Clinical trials.gov NCT06033092.

The drug's activity at the target tissue could help to define the minimal effective dose to promote cancer preventive therapy. Here we present exemestane and sex hormone concentrations within breast tissue from a pre-surgical study of alternative exemestane schedules. Postmenopausal women candidate for breast surgery for estrogen receptor-positive breast cancer were randomized to exemestane 25 mg once daily (QD), 25 mg three times/week (TIW), or 25 mg per/week (QW) for 4-6 weeks before surgery. Drug and sex hormones were analyzed from homogenized frozen tissue using a QTRAP 6500+ LC-MS/MS System. Tissue drug concentrations were detectable only in the QD arm with higher concentrations in non-malignant tissue. Estradiol was nearly suppressed in all groups in the non-malignant tissue (QD vs TIW p = .364 and QD vs QW p = .693). In contrast, a dose-response trend was observed in cancer tissue. Based on estradiol suppression in non-malignant tissue, lower exemestane schedules should be explored for breast cancer preventive therapy.

Obesity is a risk factor for postmenopausal breast cancer (BC), and evidence suggests a role for adiponectin in the relationship between obesity and BC. We investigated whether adiponectin or other biomarkers mediate the effect of body mass index (BMI) on postmenopausal BC risk in a cohort study nested in the IBIS-II Prevention Trial. We measured adiponectin, leptin, IGF-I, IGFBP-1, high-sensitivity C-reactive protein, glycemia, insulin, HOMA-IR index, and SHBG in baseline and 12-month serum samples from 123 cases and 302 matched controls in the placebo arm of the IBIS-II Prevention trial. We conducted the main mediation analysis considering baseline BMI as an exposure and the 12-month adiponectin increase as a mediator after adjustment for the Tyrer–Cuzick score and the lipid-lowering medications/supplements use. In the multivariable Cox model, both the 12-month adiponectin increase (HR, 0.60; 95%CI, 0.36–1.00) and BMI were associated with BC risk (HR, 1.05; 95%CI, 1.00–1.09), with a 40% reduction in women with a 12-month increase in adiponectin. A significantly higher cumulative hazard of BC events was observed in obese women (BMI > 30) with decreased adiponectin (p = 0.0087). No mediating effect of the adiponectin increase on the total effect of BMI on BC risk was observed (natural indirect effect: HR, 1.00; 95%CI, 0.98–1.02). Raising adiponectin levels might be an attractive target for postmenopausal BC prevention.

10510 Background: Babytam (BT), 5 mg/day for 3 years (y) is a popular choice for preventive therapy after breast DCIS/LCIS/ADH given its 10-y efficacy to prevent recurrence without adverse events (1). Here we investigated the effect of BT on serum biomarkers and their role as prognostic and predictive factors. Methods: In a phase-III trial of BT or placebo (P), 406 out of 500 women consented to blood sampling at baseline, 1 and 3 y. Serum estradiol (E2), SHBG, IGF-I, IGFBP-3 and their molar ratios were measured by CLIA. Biomarker changes were estimated with mixed-effects models for repeated measures and Incidence Rate Ratios (IRR) of the primary endpoint (invasive breast cancer or DCIS) were calculated after 5 and 10 y of follow-up with Poisson regression. Subgroup analyses were performed by interaction test. Results: There were no differences in event rates between women with blood samples and those w/out. Compared with P at 1 y, IGF-I/IGFBP-3 decreased by 0.039 (95%CI, 0.027-0.052), relative difference (rd), 22% (15-29) (p<0.001), whereas E2/SHBG increased by 2.967 (1.767-4.166), rd, 79% (47-111) in pre- and decreased by 0.121 (-1.117-0.874), rd, -9% (-84-66), in postmenopausal women on BT (p-interaction=0.001). The mean annual rate of events (x1000 PY) after 5 and 10 y follow-up according to baseline level of IGF-I/IGFBP-3 (all women) and E2/SHBG (postmenopausal). Conclusions: BT significantly lowers IGF-I/IGFBP-3 ratio, increases E2/SHBG in premenopause to a lesser extent than 20 mg/d and is effective in most subgroups regardless of baseline biomarker levels. While lower baseline IGF-I/IGFBP-3 levels were predictive of BT efficacy at 5 and 10 y, higher levels at 10 y were associated with a lower benefit of BT. E2/SHBG had a tendency to be prognostic and predictive of BT efficacy at 5 y, but softened down at 10 y. These biomarkers may help differentiate which women benefit most from BT. The role of IGF-I change as surrogate biomarker of BT effect requires further studies. 1. Lazzeroni et al: JCO 41:3116, 2023. Clinical trial information: NCT01357772 . [Table: see text]

It is well known that serum levels of oestradiol and testosterone, esp free hormone levels, influence the risk of developing breast cancer in postmenopausal women (Thomas et al 1997, Hankinson et al 1998, Kaaks et al 2005, Tin Tin et al 2021). However very little is known about how these hormone levels influence the effectiveness of aromatase inhibitors. In the IBIS-II Prevention Trial we compared anastrozole to placebo in 3864 women at high risk of breast cancer (Cuzick et al 2020). Of these women 3644 (94.3%) had a baseline blood sample. In those with a valid blood sample, 72 in the anastrozole arm and 142 in the placebo arm developed breast cancer (including DCIS) after 12.9 years of follow up (OR = 0.49, 95% CI 0.37–0.66 P< 0.0001). For each case two controls were selected, matched on age, treatment arm and follow up longer than the matching case. In these women oestradiol (E2), testosterone (Testo) and SHBG were measured by liquid chromatography – tandem mass spectroscopy, and E2/SHBG and Testo/SHBG ratios were computed to approximate free hormone levels, and analysed in quartiles. Hormone replacement therapy was not allowed during the trial, and women with use within 3 months prior to entry or outlier hormone values were excluded from these analyses. In the placebo arm higher levels of both of these ratios were associated with a higher breast cancer rate (OR per quartile 1.25 (1.04-1.59), P=0.018) for E2/SHBG and (OR per quartile 1.22 (1.02-1.47), P = 0.032) for Testo/SHBG), whereas no significant effect was seen in the anastrozole arm (OR = 1.05 (0.81-1.37), and 1.16 (0.90-1.49), resp) (Table). Neither treatment interaction was significant. Absolute numbers of cases were similar between the anastrozole and placebo arms in the lowest quartile of E2/SHBG (18 anastrozole, 22 placebo), but higher numbers were seen in the placebo arm for the other quartiles. Similar results were seen for testo/SHBG, and supportive, but weaker results were seen for these two hormones without an SHBG adjustment, SHBG and BMI at entry. Adjusting for other risk factors had no influence on these findings. Effect sizes were similar during the 5 year treatment period and thereafter. There were too few ER negative cases to compare results by receptor status. Vasomotor side effects were higher with increasing E2/SHBG levels in both arms, but no other hormone showed an effect, and no hormone level was significantly associated with gynaecologic or musculoskeletal side effects. As aromatase inhibitors effectively eliminate the production of oestradiol in postmenopausal women, these results suggest they may be more effective in postmenopausal women with high oestradiol or testosterone levels, and raise questions about their value in women in the lower quartile of serum levels for these hormones for prevention and possibly adjuvant treatment. Table: Risk of breast cancer by quartiles of the oestradiol/SHBG in ratio high risk women treated with anastrozole or placebo. Citation Format: Jack Cuzick, Kim Chu, Brian Keevil, Antony Howell, Bernardo Bonanni, Evans Gareth, Kaija Holli, Sibylle Loibl, Nicholas Zdenkowski, Steven Cummings, Mitch Dowsett. Impact of Baseline Oestradiol and Testosterone on the Preventive Effect of Anastrozole [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS10-03.

Exemestane is an effective drug to reduce breast cancer risk reaching an overall 65% reduction in breast cancer in the placebo–controlled phase III MAP.3 trial. To improve its acceptability in primary prevention programs, we are seeking the minimal effective dose. In a 3-arm presurgical trial of 4-6 weeks before breast surgery in 180 postmenopausal women with ER-positive breast cancer, we investigated the activity of alternative exemestane schedules: 25 mg per day (QD), 25 mg three times/week (TIW) or 25 mg per week (QW) and showed that in adherent participants TIW was not inferior to QD in reducing circulating estradiol (Serrano et al JAMA Oncol. doi:10.1001/jamaoncol.2023.0089). Moreover, Ki67 reduction was seen in all arms with no significant difference among arms. Here, we analyzed the concentration of sex steroids, exemestane, and its main metabolite in the cancer and adjacent non-cancerous breast tissue. Tissues samples were homogenized before liquid-liquid extraction. After reconstitution, samples were analyzed by coupling liquid chromatography with tandem mass spectrometry (Sciex QTRAP 6500, Nexera system, Shimadzu). We obtained breast cancer tissue from 93 and non-cancerous breast tissue from 117 participants to measure exemestane, 17-OH-exemestane, and sex steroids. Exemestane and 17-OH-exemestane concentrations were detectable only in the QD arm, while in TIW and QW arms levels were below the Lower Limit of Detection (< LLD). Median exemestane level was 3807 fmol/g and 17485 fmol/g and median 17-OH-exemestane level was 338 fmol/g and 1343 fmol/g in cancer and non-cancerous tissue, respectively. Interestingly, drug and its metabolite accumulated 4-5-fold in non-cancerous tissue compared to cancer tissue in the QD arm. Despite the between-arm drug concentration difference, estradiol was almost completely suppressed in all arms in the non-cancerous tissue, attaining level < LLD in QD and TIW arms, and barely detectable in QW arm. The median in the QW arm was < LLD (< LLD, interquartile range < LLD, 25.5 fmol/g) showing no differences in QD vs TIW and QD vs QW (p = 0.364 and p = 0.693 respectively). While a dose-response trend was observed in cancer tissue, estradiol level was < LLD (< LLD,52.2 fmol/g) on QD, 17.1 (< LLD, 125.3) on TIW, and 128 (< LLD, 224.8) on QW (p=0.046 QD vs TIW arms). Estrone showed a clear dose response trend among arms, whereas no differences were observed for testosterone and androstenedione for both cancer and non-cancerous tissue in all arms. The Ki-67 change was analyzed in the previous paper; here we report the data for those patients who had drug and hormones tissue concentration measured, where Ki-67 decreased in all arms: median Ki67 change from baseline was QD -8 (-10, -3), TIW -6 (-11, -2), QW -4 (-8, -1). Conclusions: Exemestane 25 mg three times a week maintains comparable activity to the standard dose on tissue estradiol suppression and Ki67 decrease. Considering the estradiol suppression in non-cancerous tissue of the lowest exemestane dose, QW might even be considered for breast cancer risk reduction in primary prevention. Further analyses are ongoing to investigate the correlation with other biomarkers including the role of polymorphic UGT2B17 genotype that could identify candidates to lower exemestane dosage. Citation Format: Davide Serrano, Harriet Johansson, Bjørn-Erik Bertelsen, Gunnar Mellgren, Parijatham Thomas, Katherine Crew, Nagi B Kumar, Debora Macis, Valentina Aristarco, Aliana Guerrieri Gonzaga, Sara Gandini, Mauro D’Amico, Tania Buttiron Webber, Irene Maria Briata, Stefano Spinaci, Viviana Galimberti, Giuseppe Viale, Lana A. Vornik, Eduardo Villar-Sanchez, Powel Brown, Brandy M Heckman-Stoddard, Eva Szabo, Bernardo Bonanni, Andrea De Censi. Exemestane and breast cancer prevention: how low can we go? Drug and biomarker tissue levels in a randomized presurgical trial on exemestane alternative dosing regimen [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS07-01.