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Supplementary methods Figure S1: The types of samples that were collected for each patient from cohort 2 (recurrence-freee patients). Figure S2: qPCR validations for Nanostring expression measurements. Figure S3: P1 miRNAs expression levels pre- and post-treatment versus normal breast tissue. Figure S4: miR-10b* expression levels. Figure S5: P1 miRNAs and their cell cycle predicted targets BUB1B and CDC25A. Figure S6: P1 scores and Ki67 levels by immunostaining. Figure S7: P1 scores in the METABRIC dataset. Figure S8: P2 miRNAs differentiate between recurrence and recurrence-free patients. Figure S9: P1 and P2 expression levels in ER-positive patients alone. Figure S10: Enrichment of EMT-induced genes in the correlated genes to P5 miRNAs. Figure S11: P5 and P6 miRNAs expression levels at post-treatment lymph nodes approach the levels at recurrence. Figure S12: Effects of the thresholds on the number of miRNAs that were classified to a pattern. Table S1: Miller and Payne grades and RCB classes Table S2: miRNAs' potential role in breast cancer from the literature Table S3: Pathway analysis; shared correlatred pathways across each pattern Table S4: Target genes for P1 miRNAs

Purpose: Molecular evolution of tumors during progression, therapy, and metastasis is a major clinical challenge and the main reason for resistance to therapy. We hypothesized that microRNAs (miRNAs) that exhibit similar variation of expression through the course of disease in several patients have a significant function in the tumorigenic process.Experimental design: Exploration of evolving disease by profiling 800 miRNA expression from serial samples of individual breast cancer patients at several time points: pretreatment, posttreatment, lymph nodes, and recurrence sites when available (58 unique samples from 19 patients). Using a dynamic approach for analysis, we identified expression modulation patterns and classified varying miRNAs into one of the eight possible temporal expression patterns.Results: The various patterns were found to be associated with different tumorigenic pathways. The dominant pattern identified an miRNA set that significantly differentiated between disease stages, and its pattern in each patient was also associated with response to therapy. These miRNAs were related to tumor proliferation and to the cell-cycle pathway, and their mRNA targets showed anticorrelated expression. Interestingly, the level of these miRNAs was lowest in matched recurrent samples from distant metastasis, indicating a gradual increase in proliferative potential through the course of disease. Finally, the average expression level of these miRNAs in the pretreatment biopsy was significantly different comparing patients experiencing recurrence to recurrence-free patients.Conclusions: Serial tumor sampling combined with analysis of temporal expression patterns enabled to pinpoint significant signatures characterizing breast cancer progression, associated with response to therapy and with risk of recurrence. Clin Cancer Res; 22(14); 3651–62. ©2016 AACR.

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Supplementary Video 2 from Real-time Imaging of Lymphogenic Metastasis in Orthotopic Human Breast Cancer

Histologic overexpression of the estrogen receptor α (ER) is a well-established prognostic marker in breast cancer. Noninvasive imaging techniques that could detect ER overexpression would be useful in a variety of settings where patients' biopsies are problematic to obtain. This study focused on developing, by in vivo MRI, strategies to measure the level of ER expression in an orthotopic mouse model of human breast cancer. Specifically, novel ER-targeted contrast agents based on pyridine-tetra-acetate-Gd(III) chelate (PTA-Gd) conjugated to 17β-estradiol (EPTA-Gd) or to tamoxifen (TPTA-Gd) were examined in ER-positive or ER-negative tumors. Detection of specific interactions of EPTA-Gd with ER were documented that could differentiate ER-positive and ER-negative tumors. In vivo competition experiments confirmed that the enhanced detection capability of EPTA-Gd was based specifically on ER targeting. In contrast, PTA-Gd acted as an extracellular probe that enhanced ER detection similarly in either tumor type, confirming a similar vascular perfusion efficiency in ER-positive and ER-negative tumors in the model. Finally, TPTA-Gd accumulated selectively in muscle and could not preferentially identify ER-positive tumors. Together, these results define a novel MRI probe that can permit selective noninvasive imaging of ER-positive tumors in vivo. Cancer Res; 71(24); 7387–97. ©2011 AACR.

Supplementary Video 3 from Real-time Imaging of Lymphogenic Metastasis in Orthotopic Human Breast Cancer

Metastatic spread to regional lymph nodes is one of the earliest events of tumor cell dissemination and presents a most significant prognostic factor for predicting survival of cancer patients. Real-time in vivo imaging of the spread of tumor cells through the lymphatic system can enhance our understanding of the metastatic process. Herein, we describe the use of in vivo fluorescence microscopy imaging to monitor the progression of lymph node metastasis as well as the course of spontaneous metastasis through the lymphatic system of orthotopic MDA-MB-231 human breast cancer tumors in severe combined immunodeficient mice. High-resolution noninvasive visualization of metastasizing cancer cells in the inguinal lymph nodes was achieved using cells expressing high levels of red fluorescent protein. Sequential imaging of these lymph nodes revealed the initial invasion of the tumor cells through the lymphatic system into the subcapsular sinuses followed by intrusion into the parenchyma of the nodes. FITC-dextran injected i.d. in the tumor area enabled simultaneous tracking of lymphatic vessels, labeled in green, and disseminated red cancer cells within these vessels. Fast snapshots of spontaneously metastasizing cells in the lymphatic vessels monitored the movement of a few tumor cells and the development of clumps clustered at lymphatic vessel junctions. Quantification of high interstitial fluid pressure (IFP) in the tumors and fast drainage rates of the FITC-dextran into the peritumoral lymphatic vessels suggested an IFP-induced intravasation into the lymphatic system. This work presents unprecedented live fluorescence images that may help to clarify the steps occurring in the course of spontaneous lymphogenic metastasis. (Cancer Res 2006; 66(16): 8037-41)

Supplementary Video 1 from Real-time Imaging of Lymphogenic Metastasis in Orthotopic Human Breast Cancer

Abnormal molecular processes occurring throughout the genome leave distinct somatic mutational patterns termed mutational signatures. Exploring the associations between mutational signatures and clinicopathological features can unravel potential mechanisms driving tumorigenic processes. We analyzed whole genome sequencing (WGS) data of tumor and peripheral blood samples from 37 primary breast cancer (BC) patients receiving neoadjuvant chemotherapy. Comprehensive clinico-pathologic features were correlated with genomic profiles and mutational signatures. Somatic mutational landscapes were highly concordant with known BC data sets. Remarkably, we observed a divergence of dominant mutational signatures in association with BC subtype. Signature 5 was overrepresented in hormone receptor positive (HR+) patients, whereas triple-negative tumors mostly lacked Signature 5, but expectedly overrepresented Signature 3. We validated these findings in a large WGS data set of BC, demonstrating dominance of Signature 5 in HR+ patients, mostly in luminal A subtype. We further investigated the association between Signature 5 and gene expression signatures, and identified potential networks, likely related to estrogen regulation. Our results suggest that the yet elusive Signature 5 represents an alternative mechanism for mutation accumulation in HR+ BC, independent of the homologous recombination repair machinery related to Signature 3. This study provides theoretical basis for further elucidating the processes promoting hormonal breast carcinogenesis.

Uveal melanoma (UM) and conjunctival melanoma (CM) are ocular malignancies that give rise to life-threatening metastases. Although local disease can often be treated successfully, it is often associated with significant vision impairment and treatments are often not effective against metastatic disease. Novel treatment modalities that preserve vision may enable elimination of small tumors and may prevent subsequent metastatic spread. Very few mouse models of metastatic CM and UM are available for research and for development of novel therapies. One of the challenges is to follow tumor growth in-vivo and to determine the right size for treatment, mainly of the posterior, choroidal melanoma. Hence, the purpose of this study was to establish a simple, noninvasive imaging tool that will simplify visualization and tumor follow-up in mouse models of CM and UM. Tumors were induced by inoculation of murine B16LS9 cells into the sub-conjunctival or the choroidal space of a C57BL/6 mouse eye under a surgical microscope. Five to ten days following injection, tumor size was assessed by Phoenix MicronIV™ image-guided Optical Coherence Tomography (OCT) imaging, which included a real-time camera view and OCT scan of the conjunctiva and the retina. In addition, tumor size was evaluated by ultrasound and histopathological examination of eye sections. Tumor growth was observed 5-9 days following sub-conjunctival or sub-retinal injection of seven-thousand or seventy-thousand cells, respectively. A clear tumor mass was detected at these regions using the MicronIV™ imaging system camera and OCT scans. Histology of eye sections confirmed the presence of tumor tissue. OCT allowed an accurate measurement of tumor size in the UM model and a qualitative assessment of tumor size in the CM model. Moreover, OCT enabled assessing the success rate of the choroidal tumor induction and importantly, predicted final tumor size already on the day of cell inoculation. In conclusion, by using a simple, non-invasive imaging tool, we were able to follow intraocular tumor growth of both CM and UM, and to define, already at the time of cell inoculation, a grading scale to evaluate tumor size. This tool may be utilized for evaluation of new mouse models for CM and UM, as well as for testing new therapies for these diseases.

Background Antrochoanal polyp (ACP) is a rare and unique unilateral nasal polyp. In contrast to diffuse primary chronic rhinosinusitis (d-CRS) the inflammatory profile of ACP in the Caucasian population have not been determined. Objective The purpose of the study is to describe and differentiate the inflammatory features of ACP compared with d-CRS and its phenotypic subgroups and hypertrophic turbinates (HT) in the Caucasian population, and compare the mast and plasma cell marker expression of each pathology. Methods A retrospective case control study of 96 patients operated on between the years 2005–2017. Nasal biopsies of ACPs, d-CRS and HTs were compared. A comparison of the different phenotypic subgroups of d-CRS was made as well. Demographics, comorbidities, and histologic and immunohistochemical (IHC) staining of mast (CD117) and plasma cell (CD138) receptor antibodies, were compared and analyzed. Results A total of 96 patients were included, consisting of 40 (41.6%) ACP, 36 (37.5%) d-CRS and 20 (20.8%) HT patients. ACPs displayed a significantly higher level of edema and intramural cysts compared to the other groups. Squamous metaplasia was demonstrated in ACP (27.5%) and d-CRS (25.6%), but not in HT. The ACP group was characterized by neutrophilic predominant infiltrates as opposed to the eosinophilic predominance in the d-CRS group, especially in eosinophilic CRS and central compartment allergic disease. ACP presented lower levels of both mast and plasma cells compared to d-CRS and HT in IHC staining. Conclusions ACP in the Caucasian population has unique features of cyst formation and edema which is compatible with its clinical presentation. It is characterized by neutrophilic predominant infiltrates and expresses lower levels of mast and plasma cells as demonstrated by IHC.

Exosomes are promising vectors for anti-tumor therapy, due to their biocompatibility, low immunogenicity, and innate ability to interact with target cells. However, promoting clinical application of exosome-based therapeutics requires elucidation of key issues, including exosome biodistribution, tumor targeting and accumulation, and the ability to overcome tumor barriers that limit the penetration of various nano-carriers and drugs. Here, we examined these parameters in exosomes derived from mesenchymal stem cells (MSC-exo) and from the A431 squamous cell carcinoma line (A431-exo), which both have potential use in cancer therapy. Using our novel technique combining gold nanoparticle (GNP) labeling of exosomes and non-invasive computed tomography imaging (CT), we longitudinally and quantitatively tracked the two intravenously-injected exosome types in A431 tumor-bearing mice. CT imaging up to 48 h and subsequent ex vivo analysis revealed tumor homing abilities of both exosome types, yet there was significantly higher tumor accumulation of MSC-exo as compared to A431-exo. Moreover, MSC-exo demonstrated the ability to penetrate the tumor and distribute throughout its bulk, while non-encapsulated GNPs remained concentrated at the tumor periphery. Histological analysis showed penetration of MSC-exo not only into the tumor tissue, but also into tumor cell cytoplasm. While the proportion of biodistribution between organs at 48 h was similar for both exosome types, more rapid clearance was indicated for A431-exo. Thus, our findings demonstrate an effect of exosome type on tumor targeting abilities and biodistribution, and suggest that MSC-exo may have superior abilities for tumor-targeted therapy.

The purpose of this study was to compare the diagnostic performance of contrast-enhanced spectral mammography (CESM) and ultrasound with that of standard digital mammography for breast cancer screening of women at intermediate risk who have dense breasts.In a retrospective cohort of 611 consecutively registered women who underwent screening CESM from 2012 to 2017, BI-RADS scores of the screening modalities were compared with actual disease status, assessed by histopathologic analysis or imaging follow-up. Sensitivity, specificity, and positive and negative predictive values were calculated.Among the 611 women included, 48.3% (295/611) had family or personal history of breast cancer, the BI-RADS breast density score was C or D in 93.1% (569/611). The mean follow-up period was 20 months. Mammography depicted 11 of 21 malignancies, sensitivity of 52.4%, specificity of 90.5% (534/590), positive predictive value of 16.4% (11/67), and negative predictive value of 98.2% (534/544). CESM depicted 19 of 21 malignancies, sensitivity of 90.5%, specificity of 76.1% (449/590), positive predictive value of 11.9% (19/160), and negative predictive value of 99.6% (449/451). Differences in sensitivity (p = 0.008) and specificity (p0.001) were statistically significant. Adjunct ultrasound revealed 73 additional suspicious findings; all were false-positive. In 39 women MRI was needed to assess screening abnormalities; two MRI-guided biopsies were performed and yielded one cancer. The incremental cancer detection rate of CESM was 13.1/1000 women (95% CI, 6.1-20.1). Of eight cancers seen only with CESM, seven were invasive (mean size, 9 mm; two of four cancers lymph-node positive).CESM was significantly more sensitive than standard digital mammography for detecting breast cancer in this screening population. No added benefit was found in the performance of ultrasound as an adjunct to CESM screens with negative results. CESM may be a valuable supplemental screening modality for women at intermediate risk who have dense breasts.

Background The main bottleneck for genomic studies of tumors is the limited availability of fresh frozen (FF) samples collected from patients, coupled with comprehensive long-term clinical follow-up. This shortage could be alleviated by using existing large archives of routinely obtained and stored Formalin-Fixed Paraffin-Embedded (FFPE) tissues. However, since these samples are partially degraded, their RNA sequencing is technically challenging. Results In an effort to establish a reliable and practical procedure, we compared three protocols for RNA sequencing using pairs of FF and FFPE samples, both taken from the same breast tumor. In contrast to previous studies, we compared the expression profiles obtained from the two matched sample types, using the same protocol for both. Three protocols were tested on low initial amounts of RNA, as little as 100 ng, to represent the possibly limited availability of clinical samples. For two of the three protocols tested, poly(A) selection (mRNA-seq) and ribosomal-depletion, the total gene expression profiles of matched FF and FFPE pairs were highly correlated. For both protocols, differential gene expression between two FFPE samples was in agreement with their matched FF samples. Notably, although expression levels of FFPE samples by mRNA-seq were mainly represented by the 3′-end of the transcript, they yielded very similar results to those obtained by ribosomal-depletion protocol, which produces uniform coverage across the transcript. Further, focusing on clinically relevant genes, we showed that the high correlation between expression levels persists at higher resolutions. Conclusions Using the poly(A) protocol for FFPE exhibited, unexpectedly, similar efficiency to the ribosomal-depletion protocol, with the latter requiring much higher (2–3 fold) sequencing depth to compensate for the relative low fraction of reads mapped to the transcriptome. The results indicate that standard poly(A)-based RNA sequencing of archived FFPE samples is a reliable and cost-effective alternative for measuring mRNA-seq on FF samples. Expression profiling of FFPE samples by mRNA-seq can facilitate much needed extensive retrospective clinical genomic studies. Electronic supplementary material The online version of this article (10.1186/s12864-018-4761-3) contains supplementary material, which is available to authorized users.

The aim of this paper is to evaluate our Unit's initial experience using the Intact breast lesion excision system as a therapeutic excision option for benign or borderline breast lesions, which otherwise would entail open operative excision. The study includes the first 111 patients who underwent therapeutic excision with the intact radiofrequency system between December 2012 and May 2016 performed at the Meirav Center for Breast Health, at the Chaim Sheba Medical Center, Ramat Gan Israel. The indications for the procedure included those patients who have benign, or atypical high risk lesions following a previous core needle biospy (CNB) who would have normally undergone conventional excision biopsy. After reviewing each case separately, we found that the use of the BLES system as a treatment device permitted the avoidance of operations that would have been otherwise indicated, in 98 cases of the total 111 in the cohort (88.3%). Thirteen cases eventually had to undergo surgical excision. In eight cases pathology was upgraded from the initial CNB to invasive or noninvasive carcinoma. Although the INTACT sysytem is most commonly used as an alternative biopsy technique for its diagnostic capabilities, this study assessed its clinical role as a definitive therapeutic excisional modality in selected cases of benign breast disease. It proved valuable in the majority of cases with avoidance of surgery where it was traditionally indicated, (98/111, 88.3%) and as definitive fibroadenoma management in a further 28 patients.

Objectives To compare the abilities of ultrasonography (US) and magnetic resonance imaging (MRI) in diagnosing silicone lymphadenopathy. Methods Consecutive patients with silicone breast implants who underwent axillary and intramammary lymph node core needle biopsies were retrospectively collected (December 2011–May 2017). Ultrasonographic examinations were analyzed for the presence of the US snowstorm sign, and MRI examinations were evaluated for the presence of the silicone signal. A pathologist reviewed all biopsied specimens. Ultrasonographic and MRI evaluations were compared to pathologic results. The sensitivity and specificity in diagnosing silicone lymphadenopathy were calculated for the snowstorm sign on US and the MRI silicone signal. Results Forty-one lymph node biopsies were included: 8 (19.5%) silicone-containing lymph nodes, 29 (70.7%) reactive nodes, and 4 (9.8%) malignant nodes. All nodes were evaluated by US, and 18 of 41 (43.9%) were evaluated by MRI. Seven of 8 (87.5%) silicone-containing nodes showed the snowstorm sign compared to none (0.0%) of the reactive or malignant nodes (P = .0001). One of 5 (20.0%) silicone-containing nodes evaluated by MRI showed the silicone signal compared to none (0.0%) of the reactive or malignant nodes (P = .278). The sensitivity and specificity of the snowstorm sign for diagnosing silicone lymphadenopathy were 87.5% and 100%, respectively, whereas those of the MRI silicone signal were 20.0% and 100%, respectively. Conclusions The US snowstorm sign is much more sensitive for silicone lymphadenopathy than the MRI silicone signal. In cases of suspected silicone lymphadenopathy, the use of US in addition to MRI should be contemplated.

The purpose of this study was to assess pathological complete response and whether it serves a surrogate for survival among patients receiving neo-adjuvant doxorubicin–cyclophosphamide followed by paclitaxel for triple-negative breast cancer with respect to BRCA1 mutation status. From a neo-adjuvant systemic therapy database of 588 breast cancer cases, 80 triple-negative cases who had undergone BRCA genotyping were identified. Logistic regression model was fitted to examine the association between BRCA1 status and pathological complete response. Survival outcomes were evaluated using Kaplan–Meier method, differences between study groups calculated by log-rank test. Thirty-four BRCA1 carriers and 43 non-carriers were identified. The BRCA1 carriers had pathological complete response rate of 68 % compared with 37 % among non-carriers, p = 0.01. Yet this did not translate into superior survival for BRCA1 carriers compared with non-carriers. No difference in relapse-free survival were noted among those with or without pathological complete response in BRCA1 carriers regardless of pathological complete response status (Log-rank p = 0.25), whereas in the non-carrier cohort, relapse-free survival was superior for those achieving pathological complete response (Log-rank p < 0.0001). Response to neo-adjuvant systemic therapy differed in BRCA1-associated triple-negative breast cancer compared with triple-negative non-carriers, with a higher rate of pathological complete response. However, compared with non-carrier triple-negative breast cancer, pathological complete response was not a surrogate for superior relapse-free survival in BRCA1 patients. Future studies using specific chemotherapy regimens may provide further improvements in outcomes.

530 Background: Tumor-infiltrating lymphocytes in breast cancer have emerged as both a prognostic and a potentially predictive immunotherapy biomarker. Advancements in artificial intelligence can extract pathology-based spatial immune fingerprints for use as treatment decision support tools. Methods: We examined 908 primary breast cancer patients with whole slide images (WSI) available from TCGA database. Digital structuring of WSIs included automated detection of lymphocytes, tumor and tumor adjacent stroma, using deep learning-based semantic segmentation. Prognosis was defined as progression free interval (PFI). A Cox Survival analysis was used to detect prognostic spatial features. We used principal component analysis (PCA) to reduce and decorrelate significant features. The resulting PCA features were used to fit the final model. The model was then validated on an independent database of WSI of breast lumpectomies, from two tertiary hospitals in Israel. Results: The analysis included 908 WSI. The average age was 58.4 years old, with a majority of early stage breast cancer (76.7%, stage I and II). The detection performance for tumor area and lymphocytes reached F1 scores of 99% and 97% respectively, in comparison to human annotation. In the Kaplan Meier (KM) analysis of 414 early stage luminal breast cancers, a high number of lymphocyte clusters (LC) and a high ratio between stromal lymphocyte density and tumor lymphocyte density (LD-S/LD-T) were significantly associated with longer PFI (p = 0.005 and p = 0.038, respectively). Based on these features, two continuous PCA features were added to the multivariate model, and remained significantly associated with PFI after adjusting for age (HR = 1.19, 95% CI 1.05-1.35; HR = 1.26 95% CI 1.03-1.55). The validation set was underpowered (n = 79) and data is still being collected. In a preliminary KM analysis of 37 early stage luminal breast cancer cases from the validation set, LD-S/LD-T was significantly associated with longer PFI (p = 0.046). Conclusions: In our study, LC and LD-S/LD-T, presumably surrogate measures of peritumoral lymphocytes, were found significantly associated with longer PFI.

Background: BRCA mutation-associated (BRCAmut) breast cancer represents a heterogeneous group displaying certain molecular features. Claudin-low breast cancers (CLBC) overlap with characteristics of BRCAmut tumors; therefore, we have investigated whether these are identical subtypes. Methods: Using public gene expression data, CLDN, CDH1, 9-cell line claudin-low predictor (9CLCLP) and PAM50 expression was evaluated in BRCAmut and BRCA wild-type (BRCAwt) breast cancer cases focusing on their possible overlap with the CLBC subtype. A separate formalin-fixed, paraffin-embedded (FFPE) cohort of 22 BRCAmut and 19 BRCAwt tumor tissues was used for immunohistochemical examination of AR, CD24, CD44, CK5/6, claudin-1, -3, -4 and -7, E-cadherin, EGFR, estrogen receptor (ER), EZH2, HER2, Ki67, p53, progesterone receptor (PgR) and vimentin expression. Results: In the data sets, CLDN1 (ROC = 0.785, p < 0.001), CDH1 (ROC = 0.785, p < 0.001), CLDN7 (ROC = 0.723, p < 0.001), CLDN3 (ROC = 0.696, p = 0.020) and CLDN4 (ROC = 0.685, p = 0.027) were expressed at higher level in BRCAmut than BRCAwt tumor tissue. The PAM50 subtype differed from the assigned immunohistochemistry (IHC)-based subtype in 30%. Based on accessible 9CLCLP predictor genes, BRCAmut breast cancer does not display the claudin-low phenotype. Utilizing FFPE samples, claudins were evidently expressed in both BRCAmut and BRCAwt cases. However, at the protein level, only claudin-3 expression was higher in BRCAmut tumors, while claudin-1, -4 and -7 and E-cadherin expression was lower compared to BRCAwt cases. A CD24low/CD44high phenotype was found in BRCAmut tumors upon comparison with BRCAwt cases (p < 0.001 and p = 0.001, respectively). Conclusions: There is a prominent correlation between the genes under focus herein and BRCA mutation status. BRCAmut tumors bear stem cell characteristics displaying a distinct cell adhesion molecule profile characterized by high expression of CDH1 and CLDN4 according to public gene expression data set analysis, and higher claudin-3 expression as detected by IHC; thus, BRCAmut breast carcinomas are not identical with the previously identified claudin-low subtype of breast cancer.

The aim of this paper is to evaluate our Unit's initial experience using the Intact breast lesion excision system as a therapeutic excision option for benign or borderline breast lesions, which otherwise would entail open operative excision. The study includes the first 111 patients who underwent therapeutic excision with the intact radiofrequency system between December 2012 and May 2016 performed at the Meirav Center for Breast Health, at the Chaim Sheba Medical Center, Ramat Gan Israel. The indications for the procedure included those patients who have benign, or atypical high risk lesions following a previous core needle biospy (CNB) who would have normally undergone conventional excision biopsy. After reviewing each case separately, we found that the use of the BLES system as a treatment device permitted the avoidance of operations that would have been otherwise indicated, in 98 cases of the total 111 in the cohort (88.3%). Thirteen cases eventually had to undergo surgical excision. In eight cases pathology was upgraded from the initial CNB to invasive or noninvasive carcinoma. Although the INTACT sysytem is most commonly used as an alternative biopsy technique for its diagnostic capabilities, this study assessed its clinical role as a definitive therapeutic excisional modality in selected cases of benign breast disease. It proved valuable in the majority of cases with avoidance of surgery where it was traditionally indicated, (98/111, 88.3%) and as definitive fibroadenoma management in a further 28 patients.

The p53 tumor suppressor serves as a crucial barrier against cancer development. In tumor cells and their progenitors, p53 suppresses cancer in a cell-autonomous manner. However, p53 also possesses non-cell-autonomous activities. For example, p53 of stromal fibroblasts can modulate the spectrum of proteins secreted by these cells, rendering their microenvironment less supportive of the survival and spread of adjacent tumor cells. We now report that epithelial tumor cells can suppress p53 induction in neighboring fibroblasts, an effect reproducible by tumor cell-conditioned medium. The ability to suppress fibroblast p53 activation is acquired by epithelial cells in the course of neoplastic transformation. Specifically, stable transduction of immortalized epithelial cells by mutant H-Ras and p53-specific short inhibitory RNA endows them with the ability to quench fibroblast p53 induction. Importantly, human cancer-associated fibroblasts are more susceptible to this suppression than normal fibroblasts. These findings underscore a mechanism whereby epithelial cancer cells may overcome the non-cell-autonomous tumor suppressor function of p53 in stromal fibroblasts.

MicroRNAs (miRNAs) belong to a class of noncoding, regulatory RNAs that is involved in oncogenesis and shows remarkable tissue specificity. Their potential for tumor classification suggests they may be used in identifying the tissue in which cancers of unknown primary origin arose, a major clinical problem. We measured miRNA expression levels in 400 paraffin-embedded and fresh-frozen samples from 22 different tumor tissues and metastases. We used miRNA microarray data of 253 samples to construct a transparent classifier based on 48 miRNAs. Two-thirds of samples were classified with high confidence, with accuracy >90%. In an independent blinded test-set of 83 samples, overall high-confidence accuracy reached 89%. Classification accuracy reached 100% for most tissue classes, including 131 metastatic samples. We further validated the utility of the miRNA biomarkers by quantitative RT-PCR using 65 additional blinded test samples. Our findings demonstrate the effectiveness of miRNAs as biomarkers for tracing the tissue of origin of cancers of unknown primary origin.

Trastuzumab in combination with adjuvant chemotherapy improves disease free survival and overall survival in HER2 over-expressing breast cancer patients. Data concerning the use of trastuzumab in the neo-adjuvant setting is limited. We aimed to compare outcome of HER2 over-expressing breast cancer patients treated with either standard chemotherapy, consisting of doxorubicin, cyclophosphamide and a taxane to outcome of patients treated with the same chemotherapy regimen with the addition of trastuzumab in concurrence with paclitaxel.We conducted a retrospective review of all consecutive HER2 over-expressing breast cancer patients treated at the participating institutions during the study period and received neo-adjuvant therapy. Allocation to trastuzumab was not based on clinical parameters and was approved only by part of the insurers. Clinical and pathological characteristics, as well as response rate and type of surgery were analyzed.Thirty seven patients received chemotherapy alone and 24 patients received chemotherapy and trastuzumab. A similar distribution of age, clinical stage and histology was noted in both groups. The rate of pathological complete response (pCR) was significantly higher among the trastuzumab-treated group compared to chemotherapy-alone group (75 vs. 24% respectively, p=0.0002). pCR in the breast was noted in 18 of 24 (75%) compared to 10 of 36 (28%, p=0.0005) and pCR in the axillary lymph nodes was noted in 19 of 20 (95%) compared to 8 of 28 (29%, p=0.0001), in the trastuzumab group compared to the chemotherapy-alone group respectively. The safety profile was similar between both groups and no clinical cardiotoxicity were noted.The addition of trastuzumab to standard chemotherapy in the neo-adjuvant setting improves pathological complete response rates in HER2 over-expressing breast cancer patients.

Metastatic spread to regional lymph nodes is one of the earliest events of tumor cell dissemination and presents a most significant prognostic factor for predicting survival of cancer patients. Real-time in vivo imaging of the spread of tumor cells through the lymphatic system can enhance our understanding of the metastatic process. Herein, we describe the use of in vivo fluorescence microscopy imaging to monitor the progression of lymph node metastasis as well as the course of spontaneous metastasis through the lymphatic system of orthotopic MDA-MB-231 human breast cancer tumors in severe combined immunodeficient mice. High-resolution noninvasive visualization of metastasizing cancer cells in the inguinal lymph nodes was achieved using cells expressing high levels of red fluorescent protein. Sequential imaging of these lymph nodes revealed the initial invasion of the tumor cells through the lymphatic system into the subcapsular sinuses followed by intrusion into the parenchyma of the nodes. FITC-dextran injected i.d. in the tumor area enabled simultaneous tracking of lymphatic vessels, labeled in green, and disseminated red cancer cells within these vessels. Fast snapshots of spontaneously metastasizing cells in the lymphatic vessels monitored the movement of a few tumor cells and the development of clumps clustered at lymphatic vessel junctions. Quantification of high interstitial fluid pressure (IFP) in the tumors and fast drainage rates of the FITC-dextran into the peritumoral lymphatic vessels suggested an IFP-induced intravasation into the lymphatic system. This work presents unprecedented live fluorescence images that may help to clarify the steps occurring in the course of spontaneous lymphogenic metastasis. (Cancer Res 2006; 66(16): 8037-41)

Objectives Elastography assesses the strain of soft tissues and is used to enhance diagnostic accuracy in evaluating breast tumors, but minimal data exist on its ability to accurately assess tumor size. This study was performed to assess the preoperative accuracy of measuring the size of biopsyproven breast cancer lesions with elastography and conventional B-mode sonography compared with the reference standard size measured by histopathologic examination. Methods Elastography and conventional B-mode sonography were performed on 69 women with histologically proven breast cancer, and tumor sizes on both modalities were recorded. These measurements were compared with the final pathologic size, which was used as the reference standard. The sizes and differences between sonographic, elastographic, and pathologic measurements were statistically tested, and an analysis of equivalence to the reference standard was performed using Bland-Altman plots. Results There was a significant difference between sizes on elastography and pathologic examination, with elastography overestimating the tumor size (P = .0187). Sonography slightly underestimated the tumor size, but this finding was not significant (P = .36). Bland-Altman plots confirmed that sonography but not elastography was an acceptable standard compared with the pathologic size. Conclusions Breast elastography but not B-mode sonography overestimates the size of breast tumors compared with the final pathologic size.

Primary malignancies of the heart and pericardium are rare. All the available data come from autopsy studies, case reports, and, in recent years, from large, specialized, single-center studies. Nevertheless, if primary malignancy is present, it may have a devastating implication for patients. Malignancies may affect heart function, also causing left-sided or right-sided heart failure. In addition, they can be responsible for embolic events or arrhythmias. Today, with the widespread use of noninvasive imaging modalities, heart tumors become evident, even as an incidental finding. A multimodality imaging approach is usually required to establish the final diagnosis. Despite the increased awareness and improved diagnostic techniques, clinical manifestations of primary malignancy of the heart and pericardium are so variable that their occurrence may still come as a surprise during surgery or autopsy. No randomized clinical trials have been carried out to determine the optimal therapy for these primary malignancies. Surgery is performed for small tumors. Chemotherapy and radiation therapy can be of help. Partial resection of large neoplasms is performed to relieve mechanical effects, such as cardiac compression or hemodynamic obstruction. Most patients present with marginally resectable or technically nonresectable disease at the time of diagnosis. It seems that orthotopic cardiac transplantation with subsequent immunosuppressive therapy may represent an option for very carefully selected patients. Early diagnosis and radical exeresis are of great importance for long-term survival of a primary cardiac malignancy. This can rarely be accomplished, and overall results are very disappointing.

Primary malignancies of the heart and pericardium are rare. All the available data come from autopsy studies, case reports, and, in recent years, from large, specialized, single‐center studies. Nevertheless, if primary malignancy is present, it may have a devastating implication for patients. Malignancies may affect heart function, also causing left‐sided or right‐sided heart failure. In addition, they can be responsible for embolic events or arrhythmias. Today, with the widespread use of noninvasive imaging modalities, heart tumors become evident, even as an incidental finding. A multimodality imaging approach is usually required to establish the final diagnosis. Despite the increased awareness and improved diagnostic techniques, clinical manifestations of primary malignancy of the heart and pericardium are so variable that their occurrence may still come as a surprise during surgery or autopsy. No randomized clinical trials have been carried out to determine the optimal therapy for these primary malignancies. Surgery is performed for small tumors. Chemotherapy and radiation therapy can be of help. Partial resection of large neoplasms is performed to relieve mechanical effects, such as cardiac compression or hemodynamic obstruction. Most patients present with marginally resectable or technically nonresectable disease at the time of diagnosis. It seems that orthotopic cardiac transplantation with subsequent immunosuppressive therapy may represent an option for very carefully selected patients. Early diagnosis and radical exeresis are of great importance for long‐term survival of a primary cardiac malignancy. This can rarely be accomplished, and overall results are very disappointing.

Forty-one patients who suffered from a Monteggia fracture and were treated between 1984 and 1993 were reviewed retrospectively using a new motion score of the elbow joint. There were 14 children and 27 adults of whom 34 were available for review. The results in children were significantly better than those of the adults. Among the adults, the final results of the patients who had a Bado Type 1 equivalent injury were significantly worse than those of the other groups, especially when associated with a fracture of the radial head. The type of fracture, open or closed, the presence of a fracture of the olecranon, and the energy level of the trauma did not have any significant prognostic value. Good correlation was shown between the new motion score and the Figgie elbow score. The Bado Type 1 equivalent injuries should be considered as a special subgroup of the Monteggia lesion, necessitating extra attention in treatment and rehabilitation, and a close followup of the patient.

Histologic overexpression of the estrogen receptor α (ER) is a well-established prognostic marker in breast cancer. Noninvasive imaging techniques that could detect ER overexpression would be useful in a variety of settings where patients' biopsies are problematic to obtain. This study focused on developing, by in vivo MRI, strategies to measure the level of ER expression in an orthotopic mouse model of human breast cancer. Specifically, novel ER-targeted contrast agents based on pyridine-tetra-acetate-Gd(III) chelate (PTA-Gd) conjugated to 17β-estradiol (EPTA-Gd) or to tamoxifen (TPTA-Gd) were examined in ER-positive or ER-negative tumors. Detection of specific interactions of EPTA-Gd with ER were documented that could differentiate ER-positive and ER-negative tumors. In vivo competition experiments confirmed that the enhanced detection capability of EPTA-Gd was based specifically on ER targeting. In contrast, PTA-Gd acted as an extracellular probe that enhanced ER detection similarly in either tumor type, confirming a similar vascular perfusion efficiency in ER-positive and ER-negative tumors in the model. Finally, TPTA-Gd accumulated selectively in muscle and could not preferentially identify ER-positive tumors. Together, these results define a novel MRI probe that can permit selective noninvasive imaging of ER-positive tumors in vivo. Cancer Res; 71(24); 7387–97. ©2011 AACR.

Activated p53 is necessary for tumor suppression. Homeodomain-interacting protein kinase-2 (HIPK2) is a positive regulator of functional p53. HIPK2 modulates wild-type p53 activity toward proapoptotic transcription and tumor suppression by the phosphorylation of serine 46. Knock-down of HIPK2 interferes with tumor suppression and sensitivity to chemotherapy. Combined administration of adriamycin and zinc restores activity of misfolded p53 and enables the induction of its proapoptotic and tumor suppressor functions in vitro and in vivo. We therefore looked for a cancer model where HIPK2 expression is low. MMTV-neu transgenic mice overexpressing HER2/neu, develop mammary tumors at puberty with a long latency, showing very low expression of HIPK2. Here we show that whereas these tumors are resistant to adriamycin treatment, a combination of adriamycin and zinc suppresses tumor growth in vivo in these mice, an effect evidenced by the histological features of the mammary tumors. The combined treatment of adriamycin and zinc also restores wild-type p53 conformation and induces proapoptotic transcription activity. These findings may open up new possibilities for the treatment of human cancers via the combination of zinc with chemotherapeutic agents, for a selected group of patients expressing low levels of HIPK2, with an intact p53. In addition, HIPK2 may serve as a new biomarker for tumor aggressiveness.

HER2 is an important prognostic and predictive marker in invasive breast cancer. It is currently assessed by immunohistochemistry for protein over-expression and by fluorescence in situ hybridization for gene amplification. The immunohistochemistry-equivocal cases (2+) are currently retested by FISH to determine eligibility for trastuzumab treatment. Retesting by FISH significantly raises the cost of patient management and sometimes delays treatment. The 4B5 is a new, FDA-approved, rabbit monoclonal antibody for HER2 testing.To examine the reliability of 4B5 IHC HER2 testing in cases found by CB11 IHC to be HER2 status equivocal.Twenty-eight invasive breast cancer cases, with an equivocal HER2 status by CB11 IHC, were retested by the 4B5 antibody as well as by FISH analysis. The scoring was performed using the same guidelines as HercepTest and was correlated with the FISH ratio.Of the original 28 CB11 clone designated equivocal cases, 14 (50%) showed negative HER2 staining using the 4B5 clone (HercepTest score 0 and 1+). Five cases (18%) proved to be positive (HercepTest score 3+) and 9 cases (32%) remained equivocal (HercepTest score 2+). The corresponding FISH ratio results showed that all 4B5 negative cases were negative by FISH testing, with a negative predictive value of 100%; 4 of 5 of the 4B5-positive cases were positive by FISH testing, with a positive predictive value of 80%. One 4B5-positive case was borderline-high (2.2 ratio) by FISH. The correlation between 4B5 IHC and FISH was statistically significant (P = 0.0013) by chi-square test.Sequential testing by 4B5 IHC could greatly reduce the need for FISH testing in cases considered HER2 equivocal by CB11 IHC.

Objectives: HER2/neu (HER2) overexpression occurs in approximately 20% of breast cancers and is associated with aggressive disease. Although a significant number of HER2-positive tumors also express hormone receptors (HR), the effects HR expression has on clinical characteristics, including response to trastuzumab among HER2-positive breast cancer, has not been elucidated yet. Methods: A retrospective analysis of consecutive metastatic HER2-positive breast cancer patients was conducted in 2 medical centers. Associations between hormone receptors expression and clinical variables, and metastatic spread pattern and survival were studied. Results: The study population included 137 metastatic HER2-positive breast cancer patients, 56 of them were HR-positive and 81 were HR-negative. No significant differences between the 2 groups were found for demographic and clinical characteristics, including age, stage at diagnosis, tumor histology, and grade. Similar response rate to trastuzumab was observed in both study groups. Significantly, longer, median, disease-free, and overall survival was noted among the HR-positive patients. Patients in the HR-negative group had significantly more liver metastases, a trend for more brain metastases, and less bone metastases. There was a strong trend for more visceral metastases in the HR-negative group. Conclusions: Our results suggest an important role for HR expression in modulating metastases predilection and disease progression in HER2-positive breast cancer.

Objective To address optimal timing of sentinel lymph node biopsy (SLNB) in breast cancer patients undergoing neoadjuvant treatment. Methods The study population included 117 patients with locally advanced cancer with clinically negative nodes treated with primary chemotherapy. Group 1 underwent SLNB and completion axillary lymph node dissection (ALND) in conjunction with lumpectomy/mastectomy, after neoadjuvant treatment (n = 31). Group 2 underwent SLNB followed by neoadjuvant therapy and subsequently surgery and completion of ALNDs (n = 58). Group 3 was treated using the same sequence as group 2, however, completion ALND was performed only for patients with positive sentinel lymph nodes (SLNs) (n = 28). Results SLN identification was lowest in group 1 compared to groups 2 and 3 (87% and 98.8% respectively; P =

The insulin-like growth factors (IGFs) play a pivotal role in breast cancer. Inherited predisposition to breast and ovarian cancer is associated with germline BRCA1/BRCA2 mutations. To evaluate the impact of BRCA1 mutations on IGF-IR gene expression, we performed an immunohistochemical analysis of IGF-IR in primary breast tumors from BRCA1 mutation carriers and non-carriers. Results obtained revealed a significant elevation in IGF-IR levels in tumors from BRCA1 mutation carriers compared with non-carriers. To assess the potential inhibitory role of BRCA1 on IGF-IR levels, we infected the BRCA1-deficient HCC1937 cell line with a BRCA1-encoding adenoviral vector. Results of Western blots showed that BRCA1 induced a large reduction in endogenous IGF-IR levels. Furthermore, results of chromatin immunoprecipitation assays indicated that the mechanism of action of BRCA1 involves interaction with Sp1, a potent transactivator of the IGF-IR gene. In conclusion, our data suggests that the IGF-IR gene is a physiologically relevant downstream target for BRCA1 action.

Tissue Microarrays (TMA) are the products of a new technology that offers rapid and simultaneous analysis of up to 1000 different archival samples. Sections from TMA blocks can be used for all different types of in situ tissue analysis including immunohistochemistry and in situ hybridization. The technology helps to implement the vast knowledge that was discovered by the human genome project and facilitates the molecular understanding of various benign and malignant processes. In this article we describe the process of TMA construction and review the main studies that applied the technology to characterizing tumors and precancerous lesions.

Cancer research has evolved at a phenomenal pace over the past two decades. Advances in genetics and molecular biology expand the knowledge gathered in the subject. Endometrial carcinoma is a common invasive malignancy in the female genital tract and presents an important diagnostic and treatment challenge. The research aims to develop new molecular tools for early diagnosis and optimal treatment of the disease and its precursors. An updated review of the molecular and genetic research is presented with correlation to the pathological and clinical characteristics of the tumor.

1023 Background: Pathological complete response (pCR) has been demonstrated to serve as a surrogate for outcome in patients receiving neo-adjuvant systemic therapy (NAT) for triple negative (TN) br...

Sheba Medical Center is a university-affiliated tertiary hospital and the largest medical center in the Middle East. Sheba's breast repository is composed of two, IRB approved, branches. The first branch, established on June 2009, is surgical, where biospecimens are collected from patients undergoing breast resection due to cancer or breast reduction for cosmetic reasons. The Second branch, established on August 2011, is the first Israeli tissue bank for breast biopsies from cancer-diagnosed women facing a neo-adjuvant therapy. Both branches collect, document and preserve biospecimens with linked clinical, pathological, demographic and other data. The surgical branch holds biospecimens of 265 donors, including: 62 donors of tumor biosamples; 93 donors of blood-only; 80 donors of blood + tumor-free tissue; 20 donors of ascites-fluid for culturing tumor cells. Molecular, biochemical and pathological QC tests, performed on representing samples, confirm that all samples are of best quality. Only 23% of samples are of tumor origin, as many breast biospecimens don't meet the rigid criteria for collection: 'Don't bank tissues under the following conditions: • When you don't have a proven biopsy of invasive breast cancer; • Where tumor size is less than 1.5-2cm; • DCIS patients. The pathologist cannot assess the specimen macroscopically; • Patients who underwent neo-adjuvant therapy. In case 'Informed Consent Form' was signed, bank blood specimens only'. Therefore, we recently established the second branch, for biopsies from cancer-diagnosed women facing a neo-adjuvant therapy. These samples are being collected when the patient arrives to mark the tumor prior to neo-adjuvant treatment. So far we collected biospecimens of 32 donors. This new concept will enable to enlarge the number and diversity of breast biospecimens that can be included in research studies. Sheba's Breast Repositories encourage studies who examine biological components and genes which may be involved in the development of tumors or genes which may affect the risk of developing cancer, the tumor's progression and its spread throughout the body as well as the response of tumor cells to various treatments.

Bone metaplasia is a rare phenomenon in the gastrointestinal tract. We present a case of a 58-year-old man who underwent laparoscopic cholecystectomy for symptoms of chronic cholecystitis. Histologic examination of the removed gallbladder revealed intramural bone metaplasia in association with chronic cholecystitis. To the best of our knowledge, such a case has not yet been reported. The clinical significance of this finding remains to be elucidated.

Abstract #5104 Background: Germline mutations in BRCA1 and BRCA2 occur in ∼ 11% of Jewish Ashkenazi women with breast cancer. BRCA1 and BRCA2 proteins are key players in the repair mechanisms of double-strand DNA breaks induced by DNA-damaging agents such as anthracyclines. Hence, inactivating mutations in BRCA1/2 genes have been postulated to result in increased chemo-sensitivity in women with breast cancer who carry these germline mutations. Neo-adjuvant therapy provides the opportunity for a rough, in vivo assessment of this notion by assessing chemotherapy (CT) sensitivity via pathological complete response (pCR) rates, in breast cancer patients, both carriers and non-carriers of deleterious BRCA1 BRCA2 mutations. In particular, response in the axillary lymph nodes to neo-adjuvant therapy is significant, as it is a surrogate marker for long-term outcome. This study assessed the pCR amongst women with BRCA1/2 mutations compared to those who are BRCA1/2 mutation negative. Methods: Retrospective evaluation of response to neo-adjuvant CT amongst consecutive women diagnosed with breast cancer who had received neo-adjuvant CT and who have all undergone genetic testing at the Oncogenetics unit, Sheba Medical Centre to define their BRCA1 BRCA2 mutational status. pCR was defined as the absence of residual invasive carcinoma in the breast and axillary lymph nodes. Only women who received anthracycline based therapy were included. Women who had received concurrent trastuzumab in the neo-adjuvant setting were excluded. Statistical analysis included Fisher's exact test for categorical variables, and student t-test for continuous variables. Results: A total of 75 women were identified who had received neo-adjuvant chemotherapy and who had undergone genetic testing. Overall, 61 were BRCA non-carriers and 14 harbored BRCA1/2 mutations (5 BRCA1 mutation carriers and 9 BRCA2 mutation carriers). There were no statistically significant differences between the BRCA1/2 positive and BRCA1/2 negative groups with regard to mean age, grade 3 histology, hormone receptor status or HER2/neu over-expression, and clinical T and N staging. Seven women received anthracycline based CT and 68 received an anthracycline and taxane regimen. No statistically significant differences existed between the two groups with regards to chemotherapy treatment protocol. Amongst women with a BRCA1/2 mutation 50% had a pCR compared with only 21% in BRCA-negative women, p=0.035. Conclusions: Women with BRCA1/2 mutations demonstrated a higher pCR compared with women who were BRCA negative. Further studies that encompass more mutation carriers with a prospective design are needed to optimize choice of chemotherapy agents in BRCA1/2 positive breast cancer patients in order to exploit the putative biological mechanisms underlying the seemingly unique chemo-sensitivity in this subset of patients. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5104.