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Background/aim Thyroid hormones (THs) stimulate breast cancer (BC) cell proliferation. We hypothesized that these hormones and the proliferative marker thymidine kinase 1 (TK1) represent the initial and final steps of the proliferative pathway, respectively. Patients and methods We measured the serum levels of thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), and free thyroxine (FT4), along with serum TK1 activity, in 144 newly diagnosed BC patients, and examined the associations between THs and proliferation in different BC receptor profiles. Results TK1 activity did not correlate with TSH (r=0.06, p=0.473) or FT4 levels (r=0.04, p=0.665), but did correlate with FT3 levels (r=0.28, p=0.001). Elevated FT3 (>6.0 pmol/l) predicted increased TK1 activity (>140 Du/l) after adjusting for age (odds ratio 4.1, p=0.014). We also found a significant association of the combined elevation of FT3 and TK1, assumed as a surrogate marker of accomplished proliferative signal, with triple-negative (TN) profile (p=0.003). The rates of combined FT3 and TK1 elevation in TN and ER-positive profiles were 20.0% and 1.8%, respectively (p=0.005). Conclusion FT3 may be involved in proliferative signaling, as measured by TK1 activity, predominately in TN breast cancer.

Introduction Traditionally, women with family history of breast and ovarian cancer, or young age at presentation, are referred to BRCA1\2 mutation testing. The growing information regarding other genes involved in predisposition to breast cancer (regardless of specific family history) and the potential impact of germline alterations on surgical and oncologic management led us to establish a model to perform a quick multigene mutation evaluation among breast cancer patients at our cancer institute. Methods All breast cancer patients that required genetic counselling (according to treating oncologist/surgeon’s judgement) were referred, after thorough explanation and signing informed-consent forms, to a 'genetic coordinator' and 'genetic nurse'. They provided each patient with a second pre-test explanation and ordered wide hereditary cancer gene panel (83-genes, Invitae). Results were given within 2-4 weeks. All patients\results were registered centrally at our center and discussed at routine Onco-genetic Multidisciplinary Board (OMB). A referral for formal genetic counselling is offered to all patients, and rapid track is arranged for patients with positive results. Results During a 12-months pilot period, 130 breast cancer patients were examined via the model, and 109 results were received; 44 patients (41%) had no abnormal findings, 50 patients (45%) had variants of uncertain clinical significance, and 15 patients (14%) were found carriers of pathogenic variants (ATM n=2, BRCA1 n=1, BRCA2 n=2, CHEK2 n=3, MUTYH n=1, PALB2 n=2, RAD50 n=1, RECQL4 n=1, TP53 n=2). Among this group, 3 patients (20%) had immediate panel-driven treatment decisions: Mastectomy to avoid radiotherapy (RAD50 carrier), oophorectomy during lumpectomy (BRCA2 carrier), and bilateral risk-reducing mastectomy instead of lumpectomy (TP53 carrier). Conclusions The creation of an Express-model for broad germline analysis allows to obtain rapid and meaningful results with impact on treatment. We believe that the Express-model should be considered as an integral tool of multimodal care of breast cancer patients. Citation Format: Albert Grinshpun, Einat Carmon, Luna Kaduri, Aviad Zick, Shiri Shkedi, Edna Shalom, Avital Granit, Liza Monas, Tamar Shaldovsky, Tamar Hamburger, Vardiella Meiner, Tamar Peretz. Express model of hereditary multigene mutation analysis in breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-14-06.

Background: Cell-free DNA (cfDNA) molecules circulating in blood are emerging as important non-invasive biomarkers for monitoring physiological and pathological processes. Tumor-derived circulating cell-free DNA (cfDNA) is present in the plasma of individuals with cancer. Assays aimed at detecting common cancer mutations in cfDNA are being developed for the detection of multiple cancer types. In breast cancer, however, such assays have failed to detect the disease at a sensitivity relevant for clinical use, in part due to the absence of multiple common mutations that can be co-detected in plasma. Unlike individual mutations that exist in only a subset of tumors, unique DNA methylation patterns are universally present in cells of a common type and therefore may be ideal biomarkers. Here we describe the detection and quantification of breast derived cfDNA, using a breast-specific DNA methylation signature. Methods: In order to identify regions of the genome with unique methylation patterns in breast epithelium, we compared genome-wide methylation profiles of normal breast tissue and breast cancer to genome-wide methylation profiles of other normal and cancerous tissues and aimed to identify CpG sites that are uniquely hypermethylated or hypomethylated in the breast. By quantifying the proportion of molecules in a sample that harbored 3 chosen breast-specific methylation sites, we could obtain an estimate of the proportion of the sample derived from breast epithelial cells. We have collected plasma prospectively from healthy women, metastatic breast cancer patients and patients with localized (stage II-III) breast cancer who were assigned to preoperative chemotherapy at our cancer institute. In the latter cohort, samples were collected before and throughout treatment until surgery. The trial was approved by the Institutional Ethics Committee and all patients gave written informed consent. Results: All three markers clearly distinguished between the plasma of healthy donors (n=64) and patients with metastatic disease (n=14, p-value Conclusion: A breast-unique epigenetic signature in circulating cell-free DNA can be used as a universal biomarker for breast cancer, to allow sensitive detection and monitoring of localized breast cancer treatment outcomes. Further research will aid to evaluate this promising and affordable method in additional clinical scenarios. Citation Format: Albert Grinshpun, Aviad Zick, Joshua Moss, Einat Carmon, Myriam Maoz, Bracha Ohana, Ofri Abraham, Lili Germansky, Benjamin Glaser, Ruth Shemer, Yuval Dor, Beatrice Uziely. Non-personalized detection of circulating breast-derived DNA for monitoring of preoperative breast cancer treatment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-10.

The purpose of this study was to assess the incremental value of preoperative breast MRI over mammography and US in depicting the accurate extent of disease in invasive duct carcinoma (IDC) compared to invasive lobular carcinoma (ILC).Retrospective analysis of pre-operative mammography, US and MRI was performed in 239 patients with either IDC (n = 193) or ILC (n = 46). Images were evaluated for solitary, multifocal or multi centric disease and compared for concordance with postsurgical pathology. Discordance was documented as either overestimation or underestimation. Two tailed paired T and Fischer's exact tests were used for analysis.Multifocality was present on pathology in 35% and 61% of patients with IDC and ILC (P.05) and multicentricity in 23% and 41% respectively (P = .84). In ILC, MRI demonstrated better concordance with pathology compared to mammography and US (89%, 44%, 49% for multifocality [P.05] and 80.5%, 63%, 71% for multicentricity [P = .3]). For IDC, concordance with pathology for all modalities was similar (65%-76%). Among discordant cases, underestimation was significantly more common for mammography and US, while MRI more frequently overestimated disease extent. MRI very rarely overestimated multifocal disease in ILC (2%).MRI demonstrates an 80% to 90% concordance rate with pathology for ILC, superior to mammography and US. The addition of MRI in IDC patients may decrease underestimation of disease extent and potentially contribute to a reduction in post-operative residual disease.

DNA methylation is a fundamental epigenetic mark that governs chromatin organization, cell identity, and gene expression. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 207 healthy tissue samples.Replicates of the same cell-type are >99.5% identical, demonstrating robustness of cell identity programs to genetic variation and environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny, and identifies methylation patterns retained since gastrulation. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hyper-methylated loci are rare and are enriched for CpG islands, polycomb targets, and CTCF binding sites, suggesting a novel role in shaping cell type-specific chromatin looping. The atlas provides an essential resource for interpretation of disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies.Summary paragraphDNA methylation, a fundamental epigenetic mark, governs chromatin organization and gene expression1, thus defining the molecular identity of cells and providing a window into developmental processes with wide-ranging physiologic and clinical ramifications. Current DNA methylation datasets have limitations, typically including only a fraction of methylation sites, many from cell lines that underwent massive changes in culture or from tissues containing unspecified mixtures of cells2–6.We present a human methylome atlas based on deep whole-genome bisulfite sequencing of 39 sorted, primary cell types and use this dataset to address fundamental questions in developmental biology, physiology and pathology. Biological replicates are >99.5% identical, demonstrating unappreciated robustness to genetic variation and environmental perturbations. Clustering recapitulates key elements of tissue ontogeny, identifying methylation patterns retained since gastrulation. Loci uniquely unmethylated in individual cell types identify novel transcriptional enhancers and are enriched for tissue-specific transcription factors binding motifs. In contrast, loci uniquely hyper-methylated in specific cell types are rare, enriched for CpG islands and polycomb targets, and overlap CTCF binding sites, suggesting a novel role in shaping cell-type-specific chromatin looping. Finally, the atlas facilitates fragment-level deconvolution of tissue and plasma methylomes across thousands of cell-type specific regions to quantify their individual components at unprecedented resolution.The human cell-type-specific methylation atlas provides an essential resource for studying gene regulation by defining cell-type-specific distal enhancers and regulators of 3D organization, for identifying pathological changes in DNA methylation, and for the interpretation of methylation-based liquid biopsies.A deep methylation atlas of 39 human cell types, sorted from healthy samplesMethylomes record developmental history of cellsThousands of novel cell type-specific methylation markersHypo-methylation uncovers cell type-specific regulatory map of distal enhancersHyper-methylation across CTCF sitesCell type-specific biomarkers facilitate fragment-level deconvolution of tissues and cfDNA

DNA methylation is a fundamental epigenetic mark that governs gene expression and chromatin organization, thus providing a window into cellular identity and developmental processes1. Current datasets typically include only a fraction of methylation sites and are often based either on cell lines that underwent massive changes in culture or on tissues containing unspecified mixtures of cells2–5. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing, allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 205 healthy tissue samples. Replicates of the same cell type are more than 99.5% identical, demonstrating the robustness of cell identity programmes to environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny and identifies methylation patterns retained since embryonic development. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hypermethylated loci are rare and are enriched for CpG islands, Polycomb targets and CTCF binding sites, suggesting a new role in shaping cell-type-specific chromatin looping. The atlas provides an essential resource for study of gene regulation and disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies.

Many experimental studies have suggested the importance of thyroid hormones in breast cancer (BC) morphogenesis. The aim of this study was to evaluate the association of thyroid hormone levels in serum of patients with primary BC with morphological presentations of the disease in pathological specimens and prognosis.We measured the serum levels of thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), and free thyroxine (FT4), along with serum thymidine kinase 1 activity and examined their relation to pathological features and prognosis of 158 patients with primary BC.We found a significant positive association of serum FT3 level with the presence of carcinoma in situ component (CIS) (p=0.032) and its size (p=0.047), with the presence (p=0.022) and the number of multifocal/multicentric tumors (MMTs) (p=0.002), as well as with increased proliferative activity in terms of serum thymidine kinase 1 (p=0.002). Moreover, we report that each 1.0 unit rise of FT3/FT4 ratio×10 was associated with an odds ratio of 1.77 (95% confidence interval=1.17-3.30, p=0.007), 1.97 (95% confidence interval=1.17-2.67, p=0.010) and 1.56 (95% confidence interval=1.02-2.37, p=0.039) for the detection of patients with CIS, MMTs and lymphovascular invasion, respectively, after adjusting for age. We did not find statistically significant associations of serum TSH level with breast cancer`s parameters. A Cox regression survival analysis identified serum FT3 level5.95 pmol/l as a risk factor for BC recurrence (relative risk=2.65, p=0.017), a finding that retained significance in a multivariate model (relative risk=2.52, p=0.027).The FT3/FT4 ratio is a valuable parameter predicting the presence of CIS, MMTs and lymphovascular invasion in pathological specimens. An elevated serum FT3 level is associated with the presence of CIS, MMTs, increased proliferative activity and poor prognosis.

BACKGROUND/AIM Silencing mediator of retinoid and thyroid receptors (SMRT) is a nuclear corepressor in thyroid and estrogen hormones pathways. The aim was to evaluate SMRT expression in relation to thyroid hormone levels and prognostic markers in breast cancer (BC). PATIENTS AND METHODS Serum and tumor tissues were obtained from 36 patients with benign breast disease (BBD) and 79 BC patients. SMRT expression was determined by immunohistochemistry. Free-triiodothyronine (FT3), free thyroxine (FT4) and thyroid-stimulating hormone (TSH) were measured in serum. RESULTS Higher FT4, lower FT3/FT4 ratio and higher expression of SMRT were found in BC compared to BBD (for all p

Summary: Differentiation events contribute to phenotypic cellular heterogeneity within tumors and influence disease progression and response to therapy. Here, we dissect mechanisms controlling intratumoral heterogeneity within triple-negative basal-like breast cancers. Tumor cells expressing the cytokeratin K14 possess a differentiation state that is associated with that of normal luminal progenitors, and K14-negative cells are in a state closer to that of mature luminal cells. We show that cells can transition between these states through asymmetric divisions, which produce one K14+ and one K14− daughter cell, and that these asymmetric divisions contribute to the generation of cellular heterogeneity. We identified several regulators that control the proportion of K14+ cells in the population. EZH2 and Notch increase the numbers of K14+ cells and their rates of symmetric divisions, and FOXA1 has an opposing effect. Our findings demonstrate that asymmetric divisions generate differentiation transitions and heterogeneity, and identify pathways that control breast cancer cellular composition. : Granit et al. study the sources of phenotypic cellular heterogeneity in triple-negative breast cancers. They find that cancer cells can undergo asymmetric divisions that produce K14+ and K14− daughters and thereby generate heterogeneity. K14+ cells possess a progenitor-associated, tumorigenic phenotype, and the authors identify regulators that control their relative numbers. Keywords: triple-negative breast cancer, basal-like breast cancer, tumor heterogeneity, asymmetric divisions, mammary progenitor cells, EZH2, Notch, FOXA1, NFIB, KLF5

Background Tumor-derived circulating cell-free DNA (cfDNA) is present in the plasma of individuals with cancer. Assays aimed at detecting common cancer mutations in cfDNA are being developed for the detection of several cancer types. In breast cancer, however, such assays have failed to detect the disease at a sensitivity relevant for clinical use, in part due to the absence of multiple common mutations that can be co-detected in plasma. Unlike individual mutations that exist only in a subset of tumors, unique DNA methylation patterns are universally present in cells of a common type and therefore may be ideal biomarkers. Here we describe the detection and quantification of breast-derived cfDNA using a breast-specific DNA methylation signature. Patients and methods We collected plasma from patients with localized breast cancer before and throughout treatment with neoadjuvant chemotherapy and surgery (N = 235 samples). Results Pretreatment breast cfDNA was detected in patients with localized disease with a sensitivity of 80% at 97% specificity. High breast cfDNA levels were associated with aggressive molecular tumor profiles and metabolic activity of the disease. During neoadjuvant chemotherapy, breast cfDNA levels decreased dramatically. Importantly, the presence of breast cfDNA towards the end of the chemotherapy regimen reflected the existence of residual disease. Conclusion We propose that breast-specific cfDNA is a universal and powerful marker for the detection and monitoring of breast cancer.

To investigate the effect of lactation on breast cancer conspicuity on dynamic contrast-enhanced (DCE) MRI in comparison with diffusion tensor imaging (DTI) parametric maps. Eleven lactating patients with 16 biopsy-confirmed pregnancy-associated breast cancer (PABC) lesions were prospectively evaluated by DCE and DTI on a 1.5-T MRI for pre-treatment evaluation. Additionally, DCE datasets of 16 non-lactating age-matched breast cancer patients were retrospectively reviewed, as control. Contrast-to-noise ratio (CNR) comprising two regions of interests of the normal parenchyma was used to assess the differences in the tumor conspicuity on DCE subtraction images between lactating and non-lactating patients, as well as in comparison against DTI parametric maps of λ1, λ2, λ3, mean diffusivity (MD), fractional anisotropy (FA), and maximal anisotropy index, λ1–λ3. CNR values of breast cancer on DCE MRI among lactating patients were reduced by 62% and 58% (p < 0.001) in comparison with those in non-lactating patients, when taking into account the normal contralateral parenchyma and an area of marked background parenchymal enhancement (BPE), respectively. Among the lactating patients, DTI parameters of λ1, λ2, λ3, MD, and λ1–λ3 were significantly decreased, and FA was significantly increased in PABC, relative to the normal lactating parenchyma ROIs. When compared against DCE in the lactating cohort, the CNR on λ1, λ2, λ3, and MD was significantly superior, providing up to 138% more tumor conspicuity, on average. Breast cancer conspicuity on DCE MRI is markedly reduced during lactation owing to the marked BPE. However, the additional application of DTI can improve the visualization and quantitative characterization of PABC, therefore possibly suggesting an additive value in the diagnostic workup of PABC. • Breast cancer conspicuity on DCE MRI has decreased by approximately 60% among lactating patients compared with non-lactating controls. • DTI-derived diffusion coefficients and the anisotropy indices of PABC lesions were significantly different than those of the normal lactating fibroglandular tissue. • Among lactating patients, breast cancer conspicuity on DTI-derived parametric maps provided up to 138% increase in contrast-to-noise ratio compared with DCE imaging.

Background: Oncotype DX is a 21-gene RT-PCR assay which quantifies the likelihood of breast cancer recurrence and the potential benefit of chemotherapy in patients with early stage, ER positive, Tamoxifen treated breast cancer. Breast cancer in BRCA carriers is considered more aggressive. The aim of our study was to evaluate whether Oncotype Dx recurrence score distribution is different in breast cancer patients with inherited BRCA mutation. Methods: The Oncotype DX assay has been used at Hadassah Medical Center since 2004 on specimens from over 450 patients. We analyzed and compared clinicopathological characteristics and Oncotype Dx recurrence scores of BRCA carriers versus non- BRCA or unknown status of BRCA patients. Results: Ten patients had validated inherited BRCA mutation, five of them are BRCA1 carriers and five BRCA2 carriers. There were no significant differences in the clinicopathological characteristics between the two groups. Oncotype Dx recurrence score distribution between low, intermediate and high risk groups was not significantly different. The mean recurrence score was 18.48 for the non- BRCA or unknown status of BRCA patients and 22.8 for the BRCA carriers patients. This difference was not statistically significant. Conclusion: Estrogen receptor positive breast cancer tumors from BRCA carriers does not display a significantly different Oncotype Dx recurrence score result distribution. These preliminary data suggest Oncotype Dx assay might be used to help tailor treatment in this subset of patients, although further follow up is needed. All patients evaluated for oncotype_DX All evaluated (except BRCA+) n = 456BRCA positiveP valueAgeMean57.4557.60.7 Median5857 Range56 (25-81)34 (42-76) T stageT167.7%70%0.9 T230.7%30% T31.6%0% Tumor sizeMean1.71.360.2 Median1.51.26 Range7.8 (0.2-8)1.3 (0.7-2) GradeGrade 1-278.8%60%0.15 Grade 321.2%40% RSLow50%40%0.57 Intermediate39.8%40% High10.2%20% RSMean18.4822.80.16 Median17.522.5 Range64 (0-64)27 (12-39) Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-05-08.

Background: Laparoscopic adjustable gastric banding (LAGB) has gained widespread acceptance. However, the technique has problems intrinsic to the material wear and tear around the port and connecting tubing that can lead to failure. Port complications are considered to be minor; however, few studies have analyzed them, and the optimal technique of port implantation and management has not been elucidated. Methods: All patients who suffered from complications involving the tubing or access-port were included in this study. Their complaints, imaging studies, operative reports and hospitalization files were retrospectively reviewed. Results: 1,272 of the patients were available for a mean follow-up period of 37 months. During this time, 91 patients (7.1%) experienced port complications that required 103 revisional operations. Of these patients, 62 had system leaks, 19 infectious problems, and 10 miscellaneous problems requiring operative correction. Overall port problems led to band removal in 6 patients, and replacement in 1 patient. Conclusion: Access-port complications after the Lap-Band® procedure are among the most common and annoying ones, and can render the device susceptible to failure. Careful surgical technique and routine use of radiologic guidance for band adjustments are the keys to avoiding complications.

Data on risperidone's efficacy and tolerability in adolescents with schizophrenia are scarce. We found only one prospective, open-label study in this population. The aim of this open-label, prospective study was to estimate the effectiveness, safety, and tolerability of risperidone treatment in adolescents with first-episode schizophrenia. Subjects were adolescent inpatients diagnosed with Diagnostic and Statistical Manual of Mental Disorders (fourth edition) first-episode schizophrenia by the Kiddie Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present Episode version. Most of the patients (10/11) were drug naive. Improvement was assessed during the first 6 weeks of treatment using the Positive and Negative Syndrome Scale (PANSS), the Brief Psychiatric Rating Scale (BPRS), and the Clinical Global Impression (CGI) scale. Side effects were monitored using the Abnormal Involuntary Movement Scale, the Simpson-Angus Scale, the Barnes Akathisia Rating Scale, and the Udvalg for Kliniske Undersogelser Side Effect Rating Scale. Eleven adolescents between 15.5 and 20 years of age (mean = 17.27, SD = 1.27 years) were included in this study. Risperidone in an average dose of 3.14 mg/day (SD = 1.60 mg/day) produced a significant improvement on the total PANSS score (28%, p < 0.01), BPRS score (30.11%, p < 0.01), and CGI-Severity score (31.36%, p < 0.01). Risperidone was ineffective in the treatment of negative signs as assessed by the PANSS. The major side effects were extrapyramidal side effects, somnolence, depression, and weight gain. In conclusion risperidone appears to be a safe, acceptably tolerated, and effective antipsychotic medication for the treatment of adolescent-onset schizophrenia.