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Epithelial-to-mesenchymal transition (EMT) is a hallmark of aggressive, mesenchymal-like high-grade serous ovarian carcinoma (HG-SOC). The SRC kinase is a key driver of cancer-associated EMT promoting adherens junction (AJ) disassembly by phosphorylation-driven internalization and degradation of AJ proteins. Here we show, that the IGF2 mRNA binding protein 1 (IGF2BP1) is up-regulated in mesenchymal-like HG-SOC and promotes SRC activation by a previously unknown protein-ligand-induced, but RNA-independent mechanism. IGF2BP1-driven invasive growth of ovarian cancer cells essentially relies on the SRC-dependent disassembly of AJs. Concomitantly, IGF2BP1 enhances ERK2 expression in a RNA-binding dependent manner. Together this reveals a post-transcriptional mechanism of interconnected stimulation of SRC/ERK signaling in ovarian cancer cells. The IGF2BP1-SRC/ERK2 axis is targetable by the SRC-inhibitor saracatinib and MEK-inhibitor selumetinib. However, due to IGF2BP1-directed stimulation only combinatorial treatment effectively overcomes the IGF2BP1-promoted invasive growth in 3D culture conditions as well as intraperitoneal mouse models. In conclusion, we reveal an unexpected role of IGF2BP1 in enhancing SRC/MAPK-driven invasive growth of ovarian cancer cells. This provides a rational for the therapeutic benefit of combinatorial SRC/MEK inhibition in mesenchymal-like HG-SOC.Graphical Abstract

Anaplastic thyroid carcinomas (ATC) are rare, but represent the most lethal malignancy of the thyroid. Selective molecular markers and drivers distinguishing ATC from other thyroid carcinomas of follicular origin remain largely unknown, limiting advances in diagnosis and treatment. In a retrospective study, we analyzed gene expression in 36 ATC, 18 poorly differentiated, 132 papillary, and 55 follicular thyroid carcinoma, as well as 124 paired and unpaired normal thyroid tissues in three independent cohorts by RNA-sequencing and immunohistochemistry. RNA-sequencing data in the test cohort suggested selective ATC protein biomarkers. Evaluation of these revealed that ATCs are characterized by the de novo expression of various testis antigens, including melanoma-associated antigen A3 (MAGEA3), but most importantly the oncofetal IGF2 mRNA binding protein 1 (IGF2BP1). Shallow whole genome sequencing essentially excluded that IGF2BP1 upregulation results from gene copy number alterations. Immunohistochemical analyses in all three tumor cohorts confirmed the selective de novo expression of IGF2BP1 protein in ATC. In sum, 75% (27/36) of all tested ATC and 0.5% (1/204) of poorly and well-differentiated thyroid carcinoma tissue samples were positive for IGF2BP1 protein. This indicates that IGF2BP1 protein expression identifies ATC with a diagnostic odds ratio of 612 (95% CI: 74.6–5021). In addition, we found that MAGEA3 is exclusively, although less consistently upregulated in ATC, presenting with an odds ratio of 411 (95% CI: 23.8–7098.7). Importantly, we provide confirmatory evidence that IGF2BP1 and MAGEA3 expression distinguishes ATC from poorly differentiated thyroid carcinoma. IGF2BP1 furthermore identified ATC foci within low-grade follicular thyroid carcinoma. In conclusion, IGF2BP1 represents the most promising single-gene marker available for ATC, followed by MAGEA3, improving on current techniques. Robust markers are essential to help distinguish this high-grade malignancy from other thyroid carcinomas, to guide surgical decision making, therapy and post-resection/therapy monitoring strategies. CA extern

Immunohistochemistry (IHC) using specific antibodies is a well-established method for the visualization of distinct cell populations. With increasing availability of suitable methods for complex tissue analyses, new demands have arisen to provide next to complex quantitative data information on protein expression, spatial distribution and cell-cell interactions in tissue sections. During the last decade, tissue preparation, fluorescent dyes, hardware imaging and software analysis were improved to solve problems concerning quantitative preciseness and tissue autofluorescence of multicolor staining. Automated cell segmentation as well as subcellular multiparameter analysis of fluorescence-based multiplexed IHC techniques, such as multispectral imaging (MSI), allows the quantification and localization of multiple proteins in the same tissue section. This technique gives us the opportunity to visualize and record the spatial relationship between different cells and is currently employed for formalin-fixed, paraffin-embedded (FFPE) samples, but has not yet been developed for calcified bone marrow (BM) biopsies. This chapter summarizes a novel protocol developed for decalcified FFPE BM samples. In addition, it discusses the technical aspects and pitfalls using this material thereby extending the use of MSI for analysis of BM malignancies. It provides an overview on the characterization and distribution of cell populations and protein expression patterns regarding their prognostic and predictive value, and their use for guidance of therapeutic decisions.

Immunohistochemistry (IHC) using specific antibodies is a well-established method for the visualization of distinct cell populations. With increasing availability of suitable methods for complex tissue analyses, new demands have arisen to provide next to complex quantitative data information on protein expression, spatial distribution and cell-cell interactions in tissue sections. During the last decade, tissue preparation, fluorescent dyes, hardware imaging and software analysis were improved to solve problems concerning quantitative preciseness and tissue autofluorescence of multicolor staining. Automated cell segmentation as well as subcellular multiparameter analysis of fluorescence-based multiplexed IHC techniques, such as multispectral imaging (MSI), allows the quantification and localization of multiple proteins in the same tissue section. This technique gives us the opportunity to visualize and record the spatial relationship between different cells and is currently employed for formalin-fixed, paraffin-embedded (FFPE) samples, but has not yet been developed for calcified bone marrow (BM) biopsies. This chapter summarizes a novel protocol developed for decalcified FFPE BM samples. In addition, it discusses the technical aspects and pitfalls using this material thereby extending the use of MSI for analysis of BM malignancies. It provides an overview on the characterization and distribution of cell populations and protein expression patterns regarding their prognostic and predictive value, and their use for guidance of therapeutic decisions.

Histomorphology has significantly changed over the last decades due to technological achievements in immunohistochemistry (IHC) for the visualization of specific proteins and in molecular pathology, particularly in the field of in situ hybridization of small oligonucleotides and amplification of DNA and RNA amplicons. With an increased availability of suitable methods, the demands regarding the observer of histomorphological slides were the supply of complex quantitative data as well as more information about protein expression and cell-cell interactions in tissue sections. Advances in fluorescence-based multiplexed IHC techniques, such as multispectral imaging (MSI), allow the quantification of multiple proteins at the same tissue section. In histopathology, it is a well-known technique for over a decade yet harboring serious problems concerning quantitative preciseness and tissue autofluorescence of multicolor staining when using formalin-fixed, paraffin-embedded (FFPE) tissue specimen. In recent years, milestones in tissue preparation, fluorescent dyes, hardware imaging, and software analysis were achieved including automated tissue segmentation (e.g., tumor vs. stroma) as well as in cellular and subcellular multiparameter analysis.This chapter covers the role that MSI plays in anatomic pathology for the analysis of FFPE tissue sections, discusses the technical aspects of MSI, and provides a review of its application in the characterization of immune cell infiltrates and beyond regarding its prognostic and predictive value and its use for guidance of clinical decisions for immunotherapeutic strategies.

Histomorphology has significantly changed over the last decades due to technological achievements in immunohistochemistry (IHC) for the visualization of specific proteins and in molecular pathology, particularly in the field of in situ hybridization of small oligonucleotides and amplification of DNA and RNA amplicons. With an increased availability of suitable methods, the demands regarding the observer of histomorphological slides were the supply of complex quantitative data as well as more information about protein expression and cell-cell interactions in tissue sections. Advances in fluorescence-based multiplexed IHC techniques, such as multispectral imaging (MSI), allow the quantification of multiple proteins at the same tissue section. In histopathology, it is a well-known technique for over a decade yet harboring serious problems concerning quantitative preciseness and tissue autofluorescence of multicolor staining when using formalin-fixed, paraffin-embedded (FFPE) tissue specimen. In recent years, milestones in tissue preparation, fluorescent dyes, hardware imaging, and software analysis were achieved including automated tissue segmentation (e.g., tumor vs. stroma) as well as in cellular and subcellular multiparameter analysis.This chapter covers the role that MSI plays in anatomic pathology for the analysis of FFPE tissue sections, discusses the technical aspects of MSI, and provides a review of its application in the characterization of immune cell infiltrates and beyond regarding its prognostic and predictive value and its use for guidance of clinical decisions for immunotherapeutic strategies.

Pre-surgical diffusion weighted imaging (DWI) is increasingly important in the context of thyroid cancer for identification of the optimal treatment strategy. It has exemplarily been shown that DWI at 3T can distinguish undifferentiated from well-differentiated thyroid carcinoma, which has decisive implications for the magnitude of surgery. This study used DWI histogram analysis of whole tumor apparent diffusion coefficient (ADC) maps. The primary aim was to discriminate thyroid carcinomas which had already gained the capacity to metastasize lymphatically from those not yet being able to spread via the lymphatic system. The secondary aim was to reflect prognostically important tumor-biological features like cellularity and proliferative activity with ADC histogram analysis. Fifteen patients with follicular-cell derived thyroid cancer were enrolled. Lymph node status, extent of infiltration of surrounding tissue, and Ki-67 and p53 expression were assessed in these patients. DWI was obtained in a 3T system using b values of 0, 400, and 800 s/mm2. Whole tumor ADC volumes were analyzed using a histogram-based approach. Several ADC parameters showed significant correlations with immunohistopathological parameters. Most importantly, ADC histogram skewness and ADC histogram kurtosis were able to differentiate between nodal negative and nodal positive thyroid carcinoma. Conclusions: histogram analysis of whole ADC tumor volumes has the potential to provide valuable information on tumor biology in thyroid carcinoma. However, further studies are warranted.

Apparent diffusion coefficient (ADC) histogram analysis has been used to some extent in cervical cancer (CC) to distinguish between low-grade and high-grade tumors. Although this differentiation is undoubtedly helpful, it would be even more crucial in the presurgical setting to determine whether a tumor already gained the potential to metastasize via the lymphatic system. So far, no studies investigated the potential of 3T ADC histogram analysis in CC to differentiate between nodal-positive and nodal-negative entities. Therefore, the principal aim of our study was to investigate the potential of 3T ADC histogram analysis to differentiate between CC with and without lymph node metastasis. The second aim was to elucidate possible differences in ADC histogram parameters between CC with limited vs. advanced tumor stages and well-differentiated vs. undifferentiated lesions. Finally, correlations of p53 expression and Ki-67 index with ADC parameters were analyzed. Eighteen female patients (mean age 55.4 years, range 32–79 years) with histopathologically confirmed cervical squamous cell carcinoma of the uterine cervix were prospectively enrolled. Tumor stages, tumor grading, status of metastatic dissemination, Ki67-index, and p53 expression were assessed in these patients. Diffusion weighted imaging (DWI) was obtained in a 3T scanner using the following b values: b0 and b1000 s/mm2. Group comparisons using Mann-Whitney U test revealed the following findings: nodal-positive CC had statistically significant lower ADC parameters (ADCmin, ADCmean, median ADC, Mode, p10, p25, p75, and p90) in comparison to nodal-negative CC (all p

Introduction: Old age and unfavorable cytogenetics are well-established adverse prognostic factors for outcome in patients (pts) with AML. Recently, high-throughput sequencing techniques have identified multiple somatically acquired driver mutations (muts) with a strong prognostic impact in AML. We explored the relative contribution of baseline molecular genetics versus a response-based sequential treatment approach on response and overall survival (OS) at two years in pts >60 years (y) with newly diagnosed AML treated within the multicenter RAS-AZIC study of the OSHO. Patients and methods: In the RAS-AZIC trial, 112 pts received priming with Azacitidine (AZA) followed by a day (d) 15 bone marrow (BM) blast count-adapted sequential treatment with AZA or intensive chemotherapy (IC). Pts with d15 blasts ≥45% received IC. If the count was Baseline BMs of 77 pts who signed the translational informed consent were analyzed by NGS using the NEOmyeloid panel of NEO New Oncology (Siemens Healthineers Company, Germany). This hybrid capture-based assay comprises 43 genes and detects point muts, small insertions and deletions in 30 genes as well as gene fusions in 18 genes. Molecular results were correlated to outcome data. Results: Median age was 70y. Secondary AML was present in 39%. Genetic risk was int-II and adverse in 25% and 27% respectively. Response on d90 was 67% (CR/CRi 58%, PR 9%). A median of 3 muts per pt were found. Mutations in DNA methylation-related-, spliceosome complex-, and chromatin-modifying-genes were 62%, 39%, and 30% respectively. Overall, muts in DNMT3A (34%), ASXL1 (25%), RUNX1 (24%), SRSF2 (23%), and TET2 (23%) were the most frequently detected (fig. 1). As expected, 72% of SRSF2 muts were associated with ASXL1 and/or RUNX1 muts. CR/CRi in the favorable and int-I 2010 ELN genetic risk groups was 81% and 57.5% in int-II and adverse risk pts. With the exception of muts in spliceosome complex-related genes, the mutational status (including the FLT3 status) seemed to have no impact on response in this cohort (table 1). In the presence of splicing factor gene muts a trend towards an inferior response of 53% compared to 77% in pts without such a mut was observed. High risk muts such as ASXL1, RUNX1, and TP53 muts which were included in the 2017 ELN adverse risk stratification were present in 20/32 (63%) of pts with favorable and int-I genetic risk. Yet, only 5/32 (16%) did not achieve a response. Similarly, CR/CRi was achieved in 7/9 (78%) pts with TP53 mut which was associated with an adverse karyotype in 8 pts. Likewise, OS tended to be similar across all molecular classes with the exception of KMT2A gene fusions with their well-known negative impact on outcome. The presence of such a fusion (n=4) was associated with a median survival time of only 8 compared to 16 months in pts without a detectable KMT2A fusion. These KMT2A gene fusions also correlated with a shorter EFS and RFS. The presence of myeloid transcription factor gene muts such as in RUNX1 (24%) and BCOR (13%) tended to be associated with an inferior EFS, while cohesin complex- (STAG2 8%), tumour suppressor-, and DNA-methylation-related gene muts with a negative RFS (table 1). Conclusions: This explorative analysis with a limited number of patients describes the pattern of genetic mutations in elderly AML treated with a response-based sequential approach that integrates epigenetic and chemotherapeutic treatments and their association with outcome. Whether such a response-based treatment mitigates the negative impact of (some) high risk mutations on outcome needs to be addressed in a much larger trial. Disclosures Jaekel: Celgene: Other: travel grant. Siebolts:Merck: Honoraria; Boehringer Ingelheim: Honoraria; MSD: Honoraria; Pfizer: Honoraria; AstraZeneca: Honoraria; Roche: Honoraria; Novartis: Honoraria; BMS: Honoraria. Pazaitis:Novartis: Consultancy. Niederwieser:Daichii: Speakers Bureau; Cellectis: Consultancy. Hänel:Amgen: Honoraria; Roche: Honoraria; Novartis: Honoraria; Takeda: Other: advisory board; Celgene: Other: advisory board. Maschmeyer:Gilead, Janssen Cilag, Astra Zeneca; BMS, Merk-Serono: Honoraria. Heukamp:NEO New Oncology GmbH: Employment. Menon:NEO New Oncology GmbH: Employment. Bertrand:NEO New Oncology GmbH: Employment. Wickenhauser:BMS: Honoraria; Merck-Serono: Honoraria; MSD: Honoraria; Böhringer: Honoraria; Novartis: Honoraria; Takeda: Honoraria; Roche: Honoraria. Brosteanu:University Leipzig: Employment. Al-Ali:Celgene: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; CTI: Honoraria.

The oncofetal IGF2 mRNA-binding protein 1 (IGF2BP1) controls the migration and invasiveness of primary as well as tumor-derived cells in vitro. Whether the protein also modulates epithelial-mesenchymal-transition (EMT), a hallmark of tumor progression involved in tumor cell dissemination, remained elusive. In this study, we reveal that IGF2BP1 enhances mesenchymal-like cell properties in tumor-derived cells by promoting the expression of the transcriptional regulators LEF1 and SLUG (SNAI2). IGF2BP1 associates with LEF1 transcripts and prevents their degradation in a 3'-UTR-dependent manner resulting in an upregulation of LEF1 expression. LEF1 promotes transcription of the mesenchymal marker fibronectin by associating with the fibronectin 1 promoter. Moreover, LEF1 enforces the synthesis of the 'EMT-driving' transcriptional regulator SNAI2. Accordingly, IGF2BP1 knockdown causes MET-like (mesenchymal-epithelial-transition) morphological changes, enhances the formation of cell-cell contacts and reduces cell migration in various mesenchymal-like tumor-derived cells. However, in epithelial-like tumor-derived cells characterized by a lack or low abundance of IGF2BP1, the protein fails to induce EMT. These findings identify IGF2BP1 as a pro-mesenchymal post-transcriptional determinant, which sustains the synthesis of 'EMT-driving' transcriptional regulators, mesenchymal markers and enhances tumor cell motility. This supports previous reports, suggesting a role of IGF2BP1 in tumor cell dissemination.

The insulin-like growth factor-2 mRNA-binding proteins 1, 2, and 3 (IGF2BP1, IGF2BP2, IGF2BP3) belong to a conserved family of RNA-binding, oncofetal proteins. Several studies have shown that these proteins act in various important aspects of cell function, such as cell polarization, migration, morphology, metabolism, proliferation and differentiation. In this review, we discuss the IGF2BP family’s role in cancer biology and how this correlates with their proposed functions during embryogenesis. IGF2BPs are mainly expressed in the embryo, in contrast with comparatively lower or negotiable levels in adult tissues. IGF2BP1 and IGF2BP3 have been found to be re-expressed in several aggressive cancer types. Control of IGF2BPs’ expression is not well understood; however, let-7 microRNAs, β-catenin (CTNNB1) and MYC have been proposed to be involved in their regulation. In contrast to many other RNA-binding proteins, IGF2BPs are almost exclusively observed in the cytoplasm where they associate with target mRNAs in cytoplasmic ribonucleoprotein complexes (mRNPs). During development, IGF2BPs are required for proper nerve cell migration and morphological development, presumably involving the control of cytoskeletal remodeling and dynamics, respectively. Likewise, IGF2BPs modulate cell polarization, adhesion and migration in tumor-derived cells. Moreover, they are highly associated with cancer metastasis and the expression of oncogenic factors (KRAS, MYC and MDR1). However, a pro-metastatic role of IGF2BPs remains controversial due to the lack of ‘classical’ in vivo studies. Nonetheless, IGF2BPs could provide valuable targets in cancer treatment with many of their in vivo roles to be fully elucidated. Electronic supplementary material The online version of this article (doi:10.1007/s00018-012-1186-z) contains supplementary material, which is available to authorized users.

OBJECTIVE: Diffusion-weighted imaging is able to reflect histopathology architecture. A novel imaging approach, namely histogram analysis, is used to further characterize lesion on MRI. To correlate histogram parameters derived from apparent diffusion coefficient (ADC) maps with histopathology parameters in muscle lymphoma. METHODS: Eight patients (mean age 64.8 years, range 45-72 years) with histopathologically confirmed muscle lymphoma were retrospectively identified. Cell count, total nucleic and average nucleic areas were estimated using ImageJ. Additionally, Ki67-index was calculated. Diffusion-weightedimaging was obtained on a 1.5 T scanner by using the b-values of 0 and 1000 s mm(−)(2). Histogram analysis was performed as a whole lesion measurement by using a custom-made Matlab-based application. RESULTS: All ADC parameters showed a good to excellent interreader variability. Cell count correlated well with ADCmean (ρ = −0.76, p = 0.03) and ADCp75 (ρ =-0.79, p = 0.02). Kurtosis and entropy correlated with average nucleic area (ρ = −0.81, p = 0.02, ρ =0.88, p = 0.007, respectively). None of the analyzed ADC parameters correlated with total nucleic area and with Ki67-index. CONCLUSION: ADC histogram analysis parameters can reflect cellularity in muscle lymphoma. ADVANCES IN KNOWLEDGE: Histogram parameters derived from ADC maps can reflect several different cellularity parameters in muscle lymphoma.

Contains fulltext : 199972.pdf (Publisher’s version ) (Open Access) Introduction: Cryptococcosis in immunocompetent adults is a rare disease in Europe, mostly induced by members of the Cryptococcus gattii species complex. The diagnosis can be challenging due to its rarity, unspecific symptoms and long symptomless latency. Case presentation: A 49-year-old woman with a three weeks history of headache was admitted to the hospital due to discrete ataxia and impaired vision. Cranial magnetic resonance imaging (MRI) showed a contrast-enhancing mass in the cerebellum. Further investigations detected a slight leukocytosis and a single subpleural nodule in the right inferior lung lobe. The cerebral lesion was surgically removed, and a direct frozen section only showed an unspecific inflammation. In the course of her admission she developed non-treatable cerebral edema and died ten days after surgical intervention. Histopathological examination of the surgical specimen and postmortem evaluation of the lung and the cerebrum demonstrated fungal elements. Molecular identification of the fungal elements in formalin-fixed paraffin-embedded tissue lead to the diagnosis of cryptococcosis induced by C. gattii sensu lato. Molecular genetic analysis identified the involved cryptococcal species as genotype AFLP6/VGII, recently described as Cryptococcus deuterogattii, which is known to be endemic to the west-coast of Canada and the USA. Additional heteroanamnestic information revealed that she had spent her holidays on Vancouver Island, Canada, two years before disease onset, indicating that infection during this stay seems to be plausible. Conclusion: Cryptococcosis due to C. deuterogattii is a rarely encountered fungal disease in Europe, not particularly associated with immunodeficiency, and infection is likely to be contracted in endemic areas. Due to its rarity, long symptomless latency, unspecific symptoms and misleading radiological features the diagnosis can be challenging. Physicians need to be aware of this differential diagnosis in immunocompetent patients, as early adequate therapy can be lifesaving.