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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.

In view of the relatively limited efficacy of immunotherapies targeting the PD-1–PD-L1 axis in triple-negative breast cancer (TNBC) and of published reports on tumor-promoting roles of TNFR2+ tumor-infiltrating lymphocytes (TNFR2+ TILs), we determined the incidence of TNFR2+ TILs in TNBC patient tumors, their association with disease outcome and relations with PD-1+ TILs. Using a cohort of treatment-naive TNBC patients with long follow-up (n = 70), we determined the presence of TNFR2+ TILs and PD-1+ TILs by immunohistochemistry. TILs (≥ 1% of cellular mass) and TNFR2+ TILs (≥ 1% of total TILs) were detected in 96% and 74% of tumors, respectively. The presence of TILs at > 5% of tumor cell mass (“Positive TILs”), as well as of positive TNFR2+ TILs (> 5%), was independently associated with good prognosis, and combination of both parameters demonstrated superior outcome relative to their lower levels. PD1+ TILs (> 5/hot spot) were detected in 63% of patients. High levels of PD-1+ TILs (> 20/hot spot) showed an unfavorable disease outcome, and in their presence, the favorable outcome of positive TNFR2+ TILs was ablated. Thus, TNFR2+ TILs are strongly connected to improved prognosis in TNBC; these findings suggest that TNFR2+ TILs have favorable effects in TNBC patients, unlike the tumor-promoting roles attributed to them in other cancer systems. Overall, our observations propose that the TNFR2+ TIL subset should not be targeted in the course of TNBC therapy; rather, its beneficial impacts may become into power when anti-PD-1 regimens—that may potentiate immune activities—are administered to TNBC patients.

SummaryDespite extensive research, internal tumor heterogeneity presents enormous challenges to achieve complete therapeutic responses. Changes in protein expression are central determinants of cancer phenotypes that reflect potential therapeutic targets. However, previous proteomic studies did not address internal heterogeneity, therefore, masked the necessary spatial resolution to achieve a comprehensive understanding of cancer complexity. Here we present the first large-scale multi-focal breast cancer proteomic study of 330 tumor regions which associated cancer cell function, pathological parameters, and spatial localization of each tumor region. We found marked internal proteomic heterogeneity even within tumors presenting homogeneous receptor expression. Additionally, analysis of the internal heterogeneity, based on coexisting receptor expression or histological patterns in single tumors, showed significant functional differences between homogeneous and heterogeneous tumors related to cancer metabolism, immunogenicity, and proliferation. We anticipate that this study will serve as a starting point towards the development of improved cancer therapy and diagnostics.

Tumor relapse as a consequence of chemotherapy resistance is a major clinical challenge in advanced stage breast tumors. To identify processes associated with poor clinical outcome, we took a mass spectrometry‐based proteomic approach and analyzed a breast cancer cohort of 113 formalin‐fixed paraffin‐embedded samples. Proteomic profiling of matched tumors before and after chemotherapy, and tumor‐adjacent normal tissue, all from the same patients, allowed us to define eight patterns of protein level changes, two of which correlate to better chemotherapy response. Supervised analysis identified two proteins of proline biosynthesis pathway, PYCR1 and ALDH18A1, that were significantly associated with resistance to treatment based on pattern dominance. Weighted gene correlation network analysis of post‐treatment samples revealed that these proteins are associated with tumor relapse and affect patient survival. Functional analysis showed that knockdown of PYCR1 reduced invasion and migration capabilities of breast cancer cell lines. PYCR1 knockout significantly reduced tumor burden and increased drug sensitivity of orthotopically injected ER‐positive tumor in vivo, thus emphasizing the role of PYCR1 in resistance to chemotherapy., Proteomic profiling of matched tumor and normal samples, associates distinct proteomic patterns with patient prognosis in breast cancer. Functional studies in vivo support the effectiveness of PYCR1 suppression in combination with chemotherapeutics in clinical settings.

This article proposes a comparative, cross-border analysis of the sources of rural combustibility around the complex frontier between Austria-Hungary, Tsarist Russia and the states that emerged out of the fringes of the Ottoman Empire, Romania, Serbia and Bulgaria, at the end of the nineteenth and beginning of the twentieth centuries. Starting from the major peasant uprising that took place in 1907 in Romania, the article seeks to account for the fledgling country's explosiveness in contrast to its neighbours, given the structural and functional similarities of the systems around the frontier. It argues that, despite these skin-deep similarities, there were vital differences as regards the initial terms of peasant emancipation and the presence of a system of checks and balances, which could curb the impositions of the great landowners onto the peasants.

This is a book about the 1907 peasant uprising in Romania but also the study of a frontier. The uprising provides the case study; the frontier provides the perspective. This moment of existential crisis in the life of the fledgling Romanian state is presented as multiple flows of information percolating and refracting internally, through the social strata, and externally, across borders. The author looks upon the three sides of the border (four, if one takes into account that Austria-Hungary was de facto two states) as distinct media with different potential for refraction and reaction.

This chapter follows the spread of the uprising in Romania and provides an account of how, within a matter of weeks, a localized incident snowballed into a country-wide rebellion threatening to remove all state authority and making a mockery of military action. It explores the ways in which peasants from distant villages got wind of the uprising elsewhere, how news circulated and what happened to this information in the process. Popular rationales for joining the uprising, newspaper reading strategies and dissemination as well as the role of popular mythologies are brought to bear on the speedy spread of violence careering from one village to the next.

This chapter proposes a study of the administrative and policing system of Romania at the time of the uprising. This, as the author argues, is vital to understanding why the uprising reached such a scale and spread in such a short time. By comparing this to the administrative systems across the border in Austria-Hungary and Tsarist Bessarabia, the chapter inaugurates the second part of the book, which tracks the reverberations of the uprising across the triple frontier at the level of administration, public opinion and diplomacy.

‘The Peasant Question’ opens up the discussion of the deep causes of the uprising analyzing from a cross-border perspective the status of the main actors of the 1907 uprising, the peasants, and their economic predicament in contrast to their peers across the triple frontier in Austria-Hungary and Tsarist Bessarabia. The transnational comparison between land tenure and the position of the peasantry in Romania and in the neighbouring provinces aims to identify those factors pertaining to legal framework and social practice that set Romania apart from its neighbours and led to different outcomes.

In this chapter, the author offers an analysis of the highest circle of news propagation and charts the coursing of information through the diplomatic structures of Austria-Hungary and Tsarist Russia, relying on correspondence between the consulates in Romania and the imperial metropolitan centres. The main concerns of the diplomats, their risk assessment advice and appraisal of the contamination potential of the uprising, as well as the measures suggested and taken, form the core of this chapter.

Background: Podoplanin (PDPN), a mucin-type transmembrane glycoprotein, is expressed in a variety of human cancer types, and contributes to tumor progression. Our goal was to evaluate PDPN expression in hepatocellular carcinoma (HCC) using both immunohistochemistry (IHC) and RNAscope in situ hybridization. Materials and Methods: Twenty patients with HCC who underwent partial hepatectomy with curative intent were retrospectively analyzed. Results: IHC gave positive results in 11 cases, while RNAscope assay for PDPN detected amplification in 16 cases. A significant association was noted between PDPN protein expression and histological tumor grade (p=0.036). Four cases that had negative PDPN results by RNAscope were also negative by IHC, while the remaining five cases with negative results by IHC were positive by RNAscope. A positive relationship was found between PDPN mRNA protein expression (p

Spectral imaging is an important method that is used for a whole spectrum of applications, but measuring very large spectral images is a challenge that so far was not achieved. We present a novel system for scanning very large spectral images of microscopy samples in a rather short time. The system captures the information while the sample is continuously being scanned on the fly. It therefore breaks the size and speed limits that resulted from existing spectral imaging methods. The spectral separation is achieved through Fourier spectroscopy by using an interferometer mounted along the optical axis (no moving parts). We describe the system and its use for pathological samples.

Introduction One of the major obstacles in breast cancer treatment is its high degree of heterogeneity and ambiguous classification. The molecular subtypes and histological subtypes are routinely defined in the clinic to determine treatment modalities; however often these distinct subtypes co-exist in single tumours and therefore lead to treatment resistance. Here, we aim to delineate the proteomic landscapes of breast cancer intratumoral diversity based on clinical-pathological parameters using mass spectrometry-based microproteomics. Material and methods Using histopathological analysis of immunostained formalin fixed paraffin embedded (FFPE) tissues, we assembled a cohort of more than 200 tumour regions comprising of different histopathological and molecular subtypes that originate from 22 patients. Cancer cell subpopulations were harvested using laser-capture microdissection (LCM) and labelled by TMT 10-plex. After protein digestion, peptides were analysed using high resolution quantitative mass spectrometry followed by computational analysis with MaxQuant. Results and discussions Topological proteomic analysis revealed the proteomic portraits that are associated with each cancer cell subpopulation, with the association to its receptor expression level and histological characteristics. We found a clear proteomic distinction between moderately and poorly differentiated regions: moderately differentiated regions were primarily segregated based on their histological subtypes such as micropapillary vs. invasive ductal carcinoma-no special type, while poorly differentiated tumour regions were distinct based on the differential expression of the oestrogen, progesterone and Her2 receptors. We identified the significantly changing proteins between these subtypes, including significant differences in metabolic enzyme profiles between co-existing subtypes. For example, we found upregulated pentose-phosphate pathway in progesterone receptor positive regions and upregulated proline/arginine biosynthesis pathways in tumour regions with micropapillary features. Conclusion These findings suggest that integrated topo-proteomic landscape of inter- and intratumoral heterogeneity can serve as a platform for deciphering the mechanisms underlying tumorigenesis, distinct metabolic profiles of cancer cell subpopulations, cancer evolution, and therapy resistance.

On 24 August 1914, Lieutenant General Artur von Bolfras warned his peer Franz Conrad von Hötzendorf that the draconian measures directed against domestic suspects were causing a lot of trouble and amounted to throwing the baby out with the bath water by fostering internal animosity. “We should not,” Bolfras insisted, “unnecessarily antagonize the population who have so far shown themselves to be loyal and ready for sacrifice beyond all expectations.” He urged in particular that Conrad should use his influence to put a stop to the excesses of the military command in Temesvár in southern Hungary, where, starting in late July 1914, hundreds of arrests were made following the partial mobilization against Serbia, thus putting into practice Conrad's professed military rationale: “better lock up one hundred people than one too few.” The initial spate of arrests and internments that accompanied the mobilization for war continued well into the autumn of 1914, snowballing into virulent state-driven repression, targeting mostly, but not only, Serbs, a situation which Josef Redlich referred to in his journal as a “race war” and a “systematic policy of extermination.” To Bolfras's letter of notification, Conrad replied that orders had been wired to Temesvár to the effect that abuses should be avoided and the military should work harmoniously with the civilian authorities, but he also signified that “we here can't do anything about individual abuses.”

The aim of this study was to analyse the expression of EGFR in newly diagnosed and recurrent glioblastoma multiforme (GBM).Our study included a total of 48 paired samples collected from 24 patients diagnosed with GBM. The intensity of EGFR cytoplasmatic staining was scored on a scale of 1-3+ (weak, intermediate or strong).We found EGFR overexpression in 23 patients (96%) with newly diagnosed GBM, while all recurrent tumours overexpressed EGFR. Ten recurrent tumours (42%) had a lower expression than their new counterpart 13 tumours (54%) had a similar expression, and only one case (2%) had increased expression on recurrence. The expression of EGFR in newly diagnosed GBM was significantly correlated with EGFR expression in recurrent tumour (p = 0.036). In addition, new GBMs with strong EGFR expression had a mean relapse-free interval of 11.5 months (p=0.017). A benefit of combined therapy was observed in the radiotherapy-plus-chemotherapy group where the average time was 11 months (p=0.011), as compared with surgery/radiotherapy alone (average time 6.8 months).The present data show that EGFR is overexpressed in paired GBMs. The discrepancies of EGFR expression between the primary tumour and the recurrence suggest heterogeneity of GBMs but also unity at relapse.

Histopathological analysis is crucial for the diagnosis of a large number of cancer types. A lot of progress has been made in the development of molecular based assays, but many of the cases still require the careful analysis of the stained tissue under a bright-field microscope and its analysis. This procedure is costly and time-consuming. We present a novel method for classification of cancer cells in lymph node images. It is based on the measurement of the spectral image of hematoxylin and eosin stained sample under the microscope and the analysis of the acquired data using state of the art machine learning techniques. The method is based on the analysis of the spectral information of the cells as well as their morphological properties. A large number of descriptors is extracted for each cell location, which are used to train a supervised classifier which discriminates between normal and cancer cells. We show that a reliable analysis can be made with detection rate (recall) of 81%-100% for the cancer class.

The purpose of this study was to test the viability of the probiotic bacteria product in a unicameral system, gastrointenstinal simulation (GIS1), for in vitro simulation of the conditions in the stomach and small intestine. The product contained a ratio of 1:1 Lactobacillus paracasei YR and Enterococcus faecium VL47 strains, in freeze-dried form and 10 g/L lactulose. The strains’ viability was evaluated under simulated gastric and small intestine conditions, in comparison with Lactobacillus johnsonii La1 strain. The experiment was performed in the presence/absence of lactulose. The strains of L. johnsonii La1 and E. faecium VL47 have shown a better resistance to bile salts and low pH, in comparison with L. paracasei YR. The survival capacity, Cs, was equal to 0 for L. paracasei YR after addition of pancreatic juice and bile salts. Losses for the other 2 strains were about 6 log CFU/mL after transit through the compartments.

Emerging mutations in the ESR1 gene that encodes for estrogen receptor (ER) have been recently associated with resistance to endocrine therapy in ER positive metastatic breast cancer patients. These mutations promote the active conformation of the receptor, conferring resistance to endocrine therapy, and were associated with shorter progression free survival. ESR1 mutations were found to rarely exist in primary tumors (~1%) but are relatively common in metastatic, endocrine therapy-resistant lesions, with an estimated prevalence of ~10-50%. Nevertheless, not much is known about the incidence of these mutations in local recurrence (breast and adjacent lymph nodes) and its clinical significance. The objective of this study is to determine the prevalence of ESR1 mutations in local recurrence of endocrine-treated breast cancer patients. To this end, we collected a cohort of breast cancer patients that had at least one local or loco-regional recurrence during or after adjuvant endocrine treatment for primary breast cancer. We analyzed the 5 most common ESR1 hotspot mutations (D538G, L536R, Y537S/N/C) using droplet digital PCR technology in 44 samples from 34 patients (some of the patients had more than one recurrence). ESR1 mutations were identified in 10/34 (29%) patients. Specifically, mutations were distributed between D538G (5/34), Y537S+ D538G (4/34) and L536R (1/34). ESR1 Mutations developed after or on Tamoxifen only treatment in 50% of the positive patients (5/10 patients) and after or on Tamoxifen + Aromatase Inhibitor (AI) treatment in the other 50% (5/10 patients). Notably, one patient developed ESR1 mutation while on neoadjuvant endocrine therapy. ESR1 mutations persisted in local recurrence samples of mutation positive patients that had more than one local recurrence and the chance of detecting a mutation in patients that had more than one recurrence was higher than in those with a single recurrence. This study demonstrates that ESR1 mutations are common in local recurrence of hormone receptor positive breast cancer. Further studies to establish the clinical significance of these mutations and their impact on survival are underway. Since local recurrences are amenable to curative therapy, the identified unexpected high prevalence of ESR1 mutations at this stage of the disease may have important consequences for choosing the optimal adjuvant treatment option for these patients. Citation Format: Adi Zundelevich, Maya Dadiani, Smadar Kahana-Edwin, Moran Gadot, Tal Sela, Irina Marin, Daniela Necula, Anya Pavlovsky, Iris Barshack, Bella Kaufman, Einav N. Gal-Yam. ESR1 mutations abundance in local recurrence of endocrine-treated breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 657.

Introduction Tumours are continuously evolving through their course of progression and treatment, a major process contributing to resistance to therapy. Increasingly, it becomes clear that unravelling the dynamics of gene expression over time, during stages of cancer progression and therapy is fundamental to interpretation of tumour evolution and resistance mechanisms. We postulate that in order to understand the emergence of resistance it is important to immensely profile matched biopsies from individual patients accompanied with their long-term clinical follow-up. Material and methods We collected triplets of archived samples from 33 individual patients that underwent neo-adjuvant (preoperative) treatment. Matched biopsies of tumour pre-treatment, post-treatment and adjacent normal epithelium as well as normal breast tissues from 6 healthy individuals were included. Full transcriptome analysis was performed by mRNA sequencing, after optimising this method for archived samples. Comprehensive clinical and pathological information was collected. A dedicated longitudinal pattern analysis method was developed to follow dynamic expression fluctuations of individual patients. Pathifier was used to calculate pathway deregulation scores. Results and discussions Principle component analysis showed clustering of the samples according to their type. Dynamic fluctuations across the 3 time-points were classified into 8 theoretical patterns, each representing a different scenario through the tumour progression and treatment stages. Genes were divided into two main types: 1. Sharing a common temporal expression pattern across most patients. These genes were associated with tumour progression pathways. 2. Genes that were divided into two or three dominant patterns and this division showed correlation with pathological response score. The dynamic pattern classification enabled to pinpoint genes associated with response that otherwise were difficult to identify using single-time point or two-time points datasets. Furthermore, the dynamics of pathway deregulation scores enabled to detect pathways that were correlated with response to therapy. Conclusion The longitudinal approach of serial sampling and analysis reveals heterogeneous dynamic behaviour across patients through the course of disease. This individual dynamics has higher sensitivity than single-time point measurements in detecting clinically relevant genes that are associated with resistance to therapy and with tumour progression.

Management of the patient with coexisting cataract and AMD presents unique challenges to the cataract surgeon, the retina specialist, and the patient. A common clinical scenario is the patient in whom both the cataract and macular pathology appear to be contributing to decreased visual acuity. As with any surgery, the expectations from cataract removal must be evaluated thoroughly and understood clearly by both the patient and the cataract surgeon. Most patients with AMD who undergo cataract surgery feel that the surgery is worthwhile, and they report improvement of visual function and quality of life. In patients with mild AMD, improvement in central visual acuity and attainment of driving vision are realistic and achievable goals. In an eye with central disciform scarring or geographic atrophy there may be potential for improvement in color discrimination, contrast, or clarity of peripheral vision. In cases of dense cataract obscuring macular detail, cataract removal may be necessary to allow for adequate biomicroscopy and angiography, especially in an eye that may be at high risk for the development of choroidal neovascularization. It is often challenging to estimate the relative impact on visual impairment made by the lens opacities and the macular changes and the benefits and risks of cataract surgery in eyes with AMD should be carefully evaluated. Is cataract surgery justified in these patients? Does cataract surgery aggravate AMD in some patients?