Real-World Data on Combined EGFR-TKI and Crizotinib Treatment for Acquired and De Novo MET Amplification in Patients with Metastatic EGFR-Mutated NSCLC.

Amplification of the mesenchymal epithelial transition (MET) gene is a mechanism of acquired resistance to epidermal growth factor receptor (EGFR)-tyrosine-kinase-inhibitors (TKIs) in over 20% of patients with advanced EGFR-mutated (EGFRm+) non-small lung cancer (NSCLC). However, it may also occur de novo in 2-8% of EGFRm+ NSCLC cases as a potential mechanism of intrinsic resistance. These patients represent a group with unmet needs, since there is no standard therapy currently approved. Several new MET inhibitors are being investigated in clinical trials, but the results are awaited. Meanwhile, as an alternative strategy, combinations of EGFR-TKIs with the MET/ALK/ROS1-TKI Crizotinib may be used in this setting, despite this use is principally off-label. Thus, we studied five of these MET amplified cases receiving EGFR-TKI and Crizotinib doublet after progression on EGFR-TKI treatment to assess the benefits and challenges related to this combination and the possible occurrence of genomic and phenotypic co-alterations. Furthermore, we compared our cases with other real-world reports on Crizotinib/EGFR-TKI combinations, which appeared effective, especially in patients with high-level MET amplification. Yet, we observed that the co-occurrence of other genomic and phenotypical alterations may affect the response to combined EGFR-TKI and Crizotinib. Finally, given the heterogeneity of MET amplification, the diagnostic methods for assessing it may be discrepant. In this respect, we observed that for optimal detection, immunohistochemistry, fluorescence in situ hybridization, and next-generation sequencing should be used together, as these methods possess different sensitivities and complement each other in characterizing MET amplification. Additionally, we addressed the issue of managing EGFR-mutated NSCLC patients with de novo MET amplification causing primary EGFR-TKI resistance. We conclude that, while data from clinical trials with new MET inhibitors are still pending, adding Crizotinib to EGFR-TKI in NSCLC patients acquiring MET amplification at progression on EGFR-TKI monotherapy is a reasonable approach, with a progression-free survival of 3-19 months.

Multi-center evaluation of the novel fully-automated PCR-based Idylla™ BRAF Mutation Test on formalin-fixed paraffin-embedded tissue of malignant melanoma.

The advent of BRAF-targeted therapies led to increased survival in patients with metastatic melanomas harboring a BRAF V600 mutation (implicated in 46-48% of malignant melanomas). The Idylla(™) System (Idylla(™)), i.e., the real-time-PCR-based Idylla(™) BRAF Mutation Test performed on the fully-automated Idylla(™) platform, enables detection of the most frequent BRAF V600 mutations (V600E/E2/D, V600K/R/M) in tumor material within approximately 90 min and with 1% detection limit. Idylla(™) performance was determined in a multi-center study by analyzing BRAF mutational status of 148 archival formalin-fixed paraffin-embedded (FFPE) tumor samples from malignant melanoma patients, and comparing Idylla(™) results with assessments made by commercial or in-house routine diagnostic methods. Of the 148 samples analyzed, Idylla(™) initially recorded 7 insufficient DNA input calls and 15 results discordant with routine method results. Further analysis learned that the quality of 8 samples was insufficient for Idylla(™) testing, 1 sample had an invalid routine test result, and Idylla(™) results were confirmed in 10 samples. Hence, Idylla(™) identified all mutations present, including 7 not identified by routine methods. Idylla(™) enables fully automated BRAF V600 testing directly on FFPE tumor tissue with increased sensitivity, ease-of-use, and much shorter turnaround time compared to existing diagnostic tests, making it a tool for rapid, simple and highly reliable analysis of therapeutically relevant BRAF mutations, in particular for diagnostic units without molecular expertise and infrastructure.

Functional Proteomic Profiling of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that comprises various disease entities, all of which share a set of common features: a lack of expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, respectively. Because of their receptor status, conventional chemotherapy remains the main therapeutic option for TNBC patients. We employed a reverse phase protein array approach (RPPA), complemented by immunohistochemistry, to quantitatively profile the activation state of 84 actionable key signaling intermediates and phosphoproteins in a set of 44 TNBC samples. We performed supervised and unsupervised approaches to proteomic data analysis to identify groups of samples sharing common characteristics that could be amenable to existing therapies. We found the heterogenous activation of multiple pathways, with PI3 K/AKT/mTOR signaling being the most common event. Some specific individualized therapeutic possibilities include the expression of oncogenic KIT in association with cytokeratin 15 and Erk1/2 positive tumors, both of which may have clinical value.

Proteomic profiling of human colon cancer cells treated with the histone deacetylase inhibitor belinostat.

The anticancer drug belinostat is a hydroxamate histone deacetylase inhibitor that has shown significant antitumour activity in various tumour models and also in clinical trials. In this study, we utilized a proteomic approach in order to evaluate the effect of this drug on protein expression in the human colon cancer cell line HCT116. Protein extracts from untreated HCT116 cells, and cells grown for 24 h in the presence of 1 and 10 muM belinostat were analysed by 2-D gel electrophoresis. Proteins were visualized by colloidal Coomassie blue staining and quantitative analysis of gel images revealed 45 unique differentially expressed proteins that were identified by LC-MSMS analysis. Among these proteins, of particular interest are the downregulated proteins nucleophosmin and stratifin, and the upregulated proteins nucleolin, gelsolin, heterogeneous nuclear ribonucleoprotein K, annexin 1, and HSP90B that all were related to the proto-oncogene proteins p53, Myc, activator protein 1, and c-fos protein. The modulation of these proteins is consistent with the observations that belinostat is able to inhibit clonogenic cell growth of HCT116 cells and the biological role of these proteins will be discussed.

Evaluation of the topoisomerase II-inactive bisdioxopiperazine ICRF-161 as a protectant against doxorubicin-induced cardiomyopathy.

Anthracycline-induced cardiomyopathy is a major problem in anti-cancer therapy. The only approved agent for alleviating this serious dose limiting side effect is ICRF-187 (dexrazoxane). The current thinking is that the ring-opened hydrolysis product of this agent, ADR-925, which is formed inside cardiomyocytes, removes iron from its complexes with anthracyclines, hereby reducing the concentration of highly toxic iron-anthracycline complexes that damage cardiomyocytes by semiquinone redox recycling and the production of free radicals. However, the 2 carbon linker ICRF-187 is also is a catalytic inhibitor of topoisomerase II, resulting in the risk of additional myelosuppression in patients receiving ICRF-187 as a cardioprotectant in combination with doxorubicin. The development of a topoisomerase II-inactive iron chelating compound thus appeared attractive. In the present paper we evaluate the topoisomerase II-inactive 3 carbon linker bisdioxopiperazine analog ICRF-161 as a cardioprotectant. We demonstrate that this compound does chelate iron and protects against doxorubicin-induced LDH release from primary rat cardiomyocytes in vitro, similarly to ICRF-187. The compound does not target topoisomerase II in vitro or in cells, it is well tolerated and shows similar exposure to ICRF-187 in rodents, and it does not induce myelosuppression when given at high doses to mice as opposed to ICRF-187. However, when tested in a model of chronic anthracycline-induced cardiomyopathy in spontaneously hypertensive rats, ICRF-161 was not capable of protecting against the cardiotoxic effects of doxorubicin. Modulation of the activity of the beta isoform of the topoisomerase II enzyme by ICRF-187 has recently been proposed as the mechanism behind its cardioprotection. This concept is thus supported by the present study in that iron chelation alone does not appear to be sufficient for protection against anthracycline-induced cardiomyopathy.

Biomarkers in tumors of the central nervous system - a review.

Until recently, diagnostics of brain tumors were almost solely based on morphology and immunohistochemical stainings for relatively unspecific lineage markers. Although certain molecular markers have been known for longer than a decade (combined loss of chromosome 1p and 19q in oligodendrogliomas), molecular biomarkers were not included in the WHO scheme until 2016. Now, the classification of diffuse gliomas rests on an integration of morphology and molecular results. Also, for many other central nervous system tumor entities, specific diagnostic, prognostic and predictive biomarkers have been detected and continue to emerge. Previously, we considered brain tumors with similar histology to represent a single disease entity. We now realize that histologically identical tumors might show alterations in different molecular pathways, and often represent separate diseases with different natural history and response to treatment. Hence, knowledge about specific biomarkers is of great importance for individualized treatment and follow-up. In this paper we review the biomarkers that we currently use in the diagnostic work-up of brain tumors.

Intrinsic resistance to EGFR-Tyrosine Kinase Inhibitors in EGFR-Mutant Non-Small Cell Lung Cancer: Differences and Similarities with Acquired Resistance.

Activating mutations in the epidermal growth factor receptor gene occur as early cancer-driving clonal events in a subset of patients with non-small cell lung cancer (NSCLC) and result in increased sensitivity to EGFR-tyrosine-kinase-inhibitors (EGFR-TKIs). Despite very frequent and often prolonged clinical response to EGFR-TKIs, virtually all advanced EGFR-mutated (EGFRM+) NSCLCs inevitably acquire resistance mechanisms and progress at some point during treatment. Additionally, 20-30% of patients do not respond or respond for a very short time (<3 months) because of intrinsic resistance. While several mechanisms of acquired EGFR-TKI-resistance have been determined by analyzing tumor specimens obtained at disease progression, the factors causing intrinsic TKI-resistance are less understood. However, recent comprehensive molecular-pathological profiling of advanced EGFRM+ NSCLC at baseline has illustrated the co-existence of multiple genetic, phenotypic, and functional mechanisms that may contribute to tumor progression and cause intrinsic TKI-resistance. Several of these mechanisms have been further corroborated by preclinical experiments. Intrinsic resistance can be caused by mechanisms inherent in EGFR or by EGFR-independent processes, including genetic, phenotypic or functional tumor changes. This comprehensive review describes the identified mechanisms connected with intrinsic EGFR-TKI-resistance and differences and similarities with acquired resistance and among clinically implemented EGFR-TKIs of different generations. Additionally, the review highlights the need for extensive pre-treatment molecular profiling of advanced NSCLC for identifying inherently TKI-resistant cases and designing potential combinatorial targeted strategies to treat them.

Differential effects of class I isoform histone deacetylase depletion and enzymatic inhibition by belinostat or valproic acid in HeLa cells.

BACKGROUND: Histone acetylation is an epigenetic modification involved in the regulation of gene expression, balanced by histone acetyl transferases and histone deacetylase (HDAC) enzymes. HDAC inhibitors (HDACi) induce growth arrest and cell death in transformed cells, and are currently in many clinical cancer trials. The transcriptional response to HDACi is complex, as is the response to HDAC isoform knockdown (KD). Here, we describe for the first time in a human cancer cell line, a transcriptional comparison of treatment by two structurally unrelated HDACi; belinostat and valproic acid with the KD of HDAC1, 2 and 3 isoforms. RESULTS: HDAC KD showed anti-proliferative effects, although to a lesser extent than HDACi treatment. Moreover, we found a 2-fold increased resistance of HDAC1 knockdown cells to belinostat, suggesting this isoenzyme as a selective target. While both HDACi treatment and individual class I HDAC KD produce significant transcriptional effects, three-times higher for HDACi, the gene-expression profiles of class I HDAC KD compared with that obtained by HDACi treatment exhibited less than 4% of altered genes in common between the two modes of inhibition. Further, cell-specific effects of HDAC KD are evident by comparison with a recent study in a different cell line. CONCLUSION: The increased resistance to belinostat in response to HDAC1 depletion indicates the possibility of using this isoform as a predictive biomarker of response to HDACi treatment. Further, the transcriptional response to chemical inhibition of HDACs is very different from that of KD of individual class I HDAC isoforms. These data suggest that the anti-tumor effect of HDACi is indeed linked to class I inhibition, but may be more complex than simply targeting individual HDAC enzymes.

Concomitant driver mutations in advanced EGFR-mutated non-small-cell lung cancer and their impact on erlotinib treatment.

BACKGROUND: Patients with EGFR-mutated non-small-cell lung cancer benefit from EGFR tyrosine kinase inhibitors (TKIs) like erlotinib. However, the efficacy may be impaired by driver mutations in other genes. METHODS: Five hundred and fourteen consecutive patients with NSCLC of all stages were tested for EGFR-mutations by cobas® EGFR Mutation Test. Fluorescent in situ hybridization (FISH) for MET-amplification, immunohistochemistry (IHC) for MET- and ALK-expression, and Next Generation Sequencing (NGS) for concomitant driver mutations were performed on EGFR-mutated tumor samples from erlotinib-treated patients. RESULTS: Thirty-six patients (7%) had EGFR-mutations, including 2 with intrinsic resistance mutation p.T790M together with the p.L858R sensitizing mutation and 1 harboring the p.G719C/S768I double-mutation. Twenty-three patients had either locally advanced or advanced disease and received first-line erlotinib-treatment. Concomitant driver mutations were found in 15/21 (71%) of NGS-analyzed TKI-treated NSCLCs, involving in 67% of cases TP53, in 13% CTNNB1, and in 7% KRAS, MET, SMAD4, PIK3CA, FGFR1, FGFR3, NRAS, DDR2, and ERBB4. No ALK-expression was found, whereas MET-overexpression and MET-amplification were observed in 5 and 4 patients, respectively. Objective responses occurred in 17/23 patients (74%), 4 did not respond (17%), and 2 harboring a SMAD4-mutation (p.R135*(stop)) and a FGFR3-mutation (p.D785fs*31), respectively, displayed mixed response with simultaneously progressing and responding tumors (8.7%). Thus, EGFR-mutated tumors harboring co-mutations were not less likely to respond. CONCLUSION: Co-mutations in other cancer-driver genes (oncogenes or tumor suppressor genes) were frequent in EGFR-mutated NSCLCs and few cases harbored concomitant activating and resistance EGFR-mutations before TKI-treatment. Most co-mutations did not impact the response to first-line erlotinib-treatment, but may represent potential additional therapeutic targets.

The PRKD1 E710D hotspot mutation is highly specific in separating polymorphous adenocarcinoma of the palate from adenoid cystic carcinoma and pleomorphic adenoma on FNA.

BACKGROUND: Polymorphous adenocarcinoma (PAC) of the palatal minor salivary glands, previously known as polymorphous low-grade adenocarcinoma, is the second most common intraoral malignant salivary gland carcinoma after adenoid cystic carcinoma (ACC) and carries an excellent prognosis. Unfortunately, PAC demonstrates cytological overlap with 2 other salivary gland tumors frequently encountered in the same location, namely ACC and pleomorphic adenoma (PA). Recently, the protein kinase D1 (PRKD1) hotspot mutation E710D was demonstrated to be specific for PAC and to be present in the majority of cases. The objective of the current study was to investigate the value of PRKD1 hotspot sequencing in identifying PAC in paired fine-needle aspiration (FNA) and surgical specimens from cases of PAC, ACC, and PA. METHODS: Paired May-Grunwald-Giemsa-stained FNA and corresponding surgical specimens were collected from 18 PAC cases, 25 ACC cases, and 21 PA cases. Both sets of specimens were subjected to dideoxynucleotide sequencing of PRKD1 exon 15, including the PRKD1 E710D hotspot. RESULTS: Of the PAC cases, approximately 50% demonstrated identical PRKD1 E710D hotspot mutations on the FNA specimen and corresponding surgical specimen. Two ACC specimens had point mutations within the sequenced region in the FNA specimen as well as the surgical specimen, but none were located in the hotspot region. None of the PA cases demonstrated PRKD1 mutations. The specificity of the PRKD1 hotspot mutation for identifying PAC among ACC and PA cases was 100% whereas the sensitivity was 50%. CONCLUSIONS: The PRKD1 E710D hotspot mutation is highly specific for identifying PAC on FNA among cases of ACC and PA, whereas the sensitivity is only modest. Alternative PRKD1 mutations exclude PAC, and are more suggestive of ACC. Cancer Cytopathol 2018;126:275-81. © 2017 American Cancer Society.

Substituted purine analogues define a novel structural class of catalytic topoisomerase II inhibitors.

By screening 1,990 compounds from the National Cancer Institute diversity set library against human topoisomerase IIalpha, we identified a novel catalytic topoisomerase II inhibitor NSC35866, a S6-substituted analogue of thioguanine. In addition to inhibiting the DNA strand passage reaction of human topoisomerase IIalpha, NSC35866 also inhibited its ATPase reaction. NSC35866 primarily inhibited DNA-stimulated ATPase activity, whereas DNA-independent ATPase activity was less sensitive to inhibition. We compared the mode of topoisomerase II ATPase inhibition induced by NSC35866 with that of 12 other substituted purine analogues of different chemical classes. The ability of thiopurines with free SH functionalities to inhibit topoisomerase II ATPase activity was completely abolished by DTT, suggesting that these thiopurines inhibit topoisomerase II ATPase activity by covalently modifying free cysteine residues. In contrast, NSC35866 as well as two O6-substituted guanine analogues, O6-benzylguanine and NU2058, could inhibit topoisomerase II ATPase activity in the presence of DTT, indicating that they have a different mechanism of inhibition. NSC35866 did not increase the level of topoisomerase II covalent cleavable complexes with DNA, indicating that it is a catalytic inhibitor and not a poison. NSC35866 was also capable of inducing a salt-stable complex of topoisomerase II on closed circular DNA. In accordance with these biochemical data, NSC35866 could antagonize etoposide-induced cytotoxicity and DNA breaks in human and murine cancer cells, confirming that NSC35866 also functions as a catalytic topoisomerase II inhibitor in cells.

Lacrimal gland ductal carcinomas: Clinical, Morphological and Genetic characterization and implications for targeted treatment.

PURPOSE: Ductal carcinomas (DCs) of the lacrimal gland are very rare but aggressive malignancies. We investigated DC of the lacrimal gland for potentially clinically actionable targets in the search for new therapeutic options. METHODS: Case 1: A 77-year-old man, presented with diplopia and xerophtalmia; case 2: A 53-year-old man, presented with headache, proptosis and chemosis and case 3: A 73-year-old man, presenting with chemosis and a corneal abscess. All three cases were characterized morphologically including immunohistochemistry and genetically with fluorescence in situ hybridization (FISH) and one case with next-generation sequencing (NGS) of cancer relevant genes. RESULTS: Cases 1 and 3 were composed of large, rounded, irregular cystic nodules of carcinoma cells with prominent central comedonecrosis, whereas case 2 had a scirrhous morphology. High expression of CK7, CK19, EMA, p53 and HER2 was characteristic for all three tumours. Androgen receptor was intensely positive in case 1, in scattered cells in case 2 and negative in case 3, whereas oestrogen and progesterone receptor were consistently negative. Genetically, a hemizygous deletion and a point mutation in PTEN were identified in case 1, whereas HER2 amplification was found in cases 2 and 3. CONCLUSION: This study identified a spectrum of genetic events and pattern of protein expression in DC of the lacrimal gland similar to a subset of carcinomas of the breast and ductal carcinomas of the salivary glands. For therapeutic purposes, aberrations in several components of especially the HER2 signalling pathway could alleviate the effect of HER2-directed therapy illustrating an inadequacy of isolated HER2 testing.

Heterogeneous resistance mechanisms in an EGFR exon 19-mutated non-small cell lung cancer patient treated with erlotinib: Persistent FGFR3-mutation, localized transformation to EGFR-mutated SCLC, and acquired T790M EGFR-mutation.

Patients with epidermal growth factor receptor (EGFR) gene-mutated non-small cell lung cancer (NSCLC) obtain substantial clinical benefit from EGFR tyrosine-kinase inhibitors (TKIs), but will ultimately develop TKI-resistance resulting in median progression-free survival of 9-15 months during first-line TKI-therapy. However, type and timing of TKI-resistance cannot be predicted and several mechanisms may simultaneously/subsequently occur during TKI-treatment. In this respect, we present a 49 year-old Caucasian male ex-smoker with metastatic pulmonary adenocarcinoma (ADC) that concomitantly harbored an EGFR exon 19-mutation (p.E746_A750delELREA) and a previously unreported 2bp frame-shift microdeletion in the fibroblast growth factor receptor 3 (FGFR3; p.D785fs*31) gene. Interestingly, FGFR3-mutations have previously been described in other cancer types of Caucasian patients and may represent an alternative pathway to EGFR-signaling. The patient received first-line erlotinib but after only 7 weeks showed metastatic pleural effusion, in which transformation to small cell lung cancer (SCLC) that retained the EGFR- and FGFR3-mutations was identified. Consequently, standard carboplatin-etoposide regimen for SCLC combined with erlotinib continuation was implemented obtaining significant objective response. However, after completing 6 cycles of this combination, new pulmonary and hepatic metastases appeared and showed persistence of the original EGFR- and FGFR3-mutated ADC phenotype together with acquisition of the erlotinib-resistant T790M EGFR-mutation. The patient rapidly deteriorated and deceased. Thus, this advanced EGFR-mutated NSCLC displayed very rapid onset and heterogeneous genetic and phenotypic mechanisms of TKI-resistance occurring at different times and locations of metastatic disease: concomitant FGFR3-mutation before and during TKI-treatment as potential intrinsic mechanism for the rapid progression; transformation to SCLC at first progression during TKI-therapy; acquired T790M EGFR-mutation at second progression. Our case also underlines that, when achievable, rebiopsies of progressive sites during TKI-treatment are important for identifying heterogeneous histopathological and molecular resistance mechanisms and better defining possible treatment modifications.

The interleukin-6/Janus kinase/STAT3 pathway in pleomorphic adenoma and carcinoma ex pleomorphic adenoma of the lacrimal gland.

PURPOSE: Pleomorphic adenoma (PA) is the most common tumour of the lacrimal gland, but very little is known about its biology. It has a tendency to recur and an ability to transform into the high-grade malignancy carcinoma ex pleomorphic adenoma (ca-ex-PA), which is also largely unexplored. In this study, we examine the expression of the interleukin-6/Janus kinase/STAT3 (IL-6/JAK/STAT3) pathway components in PA and ca-ex-PA. METHODS: Sixteen PAs and two ca-ex-PAs were examined with immunohistochemistry. Seven PAs were subjected to microdissection and subsequent qPCR. RESULTS: The IL-6/JAK/STAT3 pathway was overexpressed in PA compared to normal lacrimal gland. Overexpression of phosphorylated JAK1 (p-JAK1) and cyclin D1 was significantly overexpressed in ductal cells compared with myoepithelial cells in PA. A shift from p-JAK1 to p-JAK2 and p-Tyk2 overexpression was seen between PA and ca-ex-PA, combined with a high p-STAT3 expression in the latter. CONCLUSION: The IL-6/JAK/STAT3 pathway is overexpressed in PA, and this overexpression was even more pronounced in ca-ex-PA, with a shift in the JAKs mediating STAT3 phosphorylation. Future studies are needed to clarify whether PA and ca-ex-PA could be treated with targeted therapy directed against components of the IL-6/JAK/STAT3 pathway.

Multi-Center Evaluation of the Fully Automated PCR-Based Idylla™ KRAS Mutation Assay for Rapid KRAS Mutation Status Determination on Formalin-Fixed Paraffin-Embedded Tissue of Human Colorectal Cancer.

Since the advent of monoclonal antibodies against epidermal growth factor receptor (EGFR) in colorectal cancer therapy, the determination of RAS mutational status is needed for therapeutic decision-making. Most prevalent in colorectal cancer are KRAS exon 2 mutations (40% prevalence); lower prevalence is observed for KRAS exon 3 and 4 mutations (6%) and NRAS exon 2, 3, and 4 mutations (5%). The Idylla™ KRAS Mutation Test on the molecular diagnostics Idylla™ platform is a simple (<2 minutes hands-on time), highly reliable, and rapid (approximately 2 hours turnaround time) in vitro diagnostic sample-to-result solution. This test enables qualitative detection of 21 mutations in codons 12, 13, 59, 61, 117, and 146 of the KRAS oncogene being clinically relevant according to the latest clinical guidelines. Here, the performance of the Idylla™ KRAS Mutation Assay, for Research Use Only, was assessed on archived formalin-fixed paraffin-embedded (FFPE) tissue sections by comparing its results with the results previously obtained by routine reference approaches for KRAS genotyping. In case of discordance, samples were assessed further by additional methods. Among the 374 colorectal cancer FFPE samples tested, the overall concordance between the Idylla™ KRAS Mutation Assay and the confirmed reference routine test results was found to be 98.9%. The Idylla™ KRAS Mutation Assay enabled detection of 5 additional KRAS-mutated samples not detected previously with reference methods. As conclusion the Idylla™ KRAS Mutation Test can be applied as routine tool in any clinical setting, without needing molecular infrastructure or expertise, to guide the personalized treatment of colorectal cancer patients.

Medullary carcinoma of the colon: can the undifferentiated be differentiated?

Medullary carcinoma of the colon is a rare variant of colorectal cancer claimed to have a more favorable prognosis than conventional adenocarcinomas. The histopathologic appearance may be difficult to distinguish from poorly differentiated adenocarcinoma. The study aimed to evaluate the diagnostic interobserver agreement and to characterize the immunohistochemical and molecular differences between these two subgroups. Fifteen cases initially classified as medullary carcinoma and 30 cases of poorly differentiated adenocarcinomas were included. Two pathologists reviewed the slides independently without knowledge of the original diagnosis and subgrouped the tumors into the two entities. Agreement was reached in 31 of 45 cases (69 %) with kappa = 0.32. An extensive immunohistochemical panel was performed, and KRAS, NRAS, and BRAF mutational status was assessed. Of the 31 cases with diagnostic agreement, the expression of only MLH-1 along with corresponding expression of PMS-2 differed significantly (p = 0.04). A high rate of BRAF mutations was detected in both subgroups without significant differences. Expression of MLH-1 was superior in dividing the tumors into two separate entities with significant differences in CK20 (p = 0.005) expression and in the rate of BRAF mutations (p = 0.0035). In conclusion, medullary carcinomas of the colon are difficult to discriminate from poorly differentiated adenocarcinoma even with the help of immunohistochemical and molecular analyses. This raises the question whether these morphological subtypes should be maintained or whether an alternative classification of poorly differentiated colorectal adenocarcinomas based on MLH-1 status rather than morphology should be suggested.

Activation of the interleukin-6/Janus kinase/STAT3 pathway in pleomorphic adenoma of the parotid gland.

The interleukin-6 (IL-6)/Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway is of crucial importance in promoting tumorigenesis in several malignant tumors but may also be active in benign tumors, e.g., of pleomorphic adenoma (PA). In this study we characterize the expression of the pathway components with immunohistochemistry and selected mRNAs and microRNAs (miRNAs) regulated by this pathway in isolated duct- and myoepithelial cells in PA. 46 PAs were immunostained and 10 of these were used for in situ hybridization (ISH). Six frozen specimens were analyzed using reverse transcription-polymerase chain reaction (RT-PCR). Using immunohistochemistry, IL-6, JAK1, JAK2 and STAT3 were detected significantly more frequently in PA cells than in cells from normal salivary gland tissue. Using RT-PCR cyclin D1, fibroblast growth factor 2, and p21 were found to be overexpressed while matrix metallopeptidase 9 was detected at low levels in PA compared to normal salivary gland. ISH showed significant overexpression of miR-181b in PA, while miR-21 was undetectable in PA and normal tissue. Overexpression of the pathway components and its mRNA and miRNA products provide important clues regarding the growth of PAs. Our findings brings us one step closer to targeted treatment of this tumor entity, although in vitro studies are warranted to confirm this.

Methylation-associated Silencing of microRNA-126 and its Host Gene EGFL7 in Malignant Pleural Mesothelioma.

BACKGROUND/AIM: We recently reported that miR-126 is down-regulated in malignant pleural mesothelioma (MPM) and can be combined into a 4-microRNA-classifier that can accurately diagnose MPM with high sensitivity and specificity. Herein we analyzed the epigenetic regulation of miR-126 and its host gene EGF-like domain, multiple 7 (EGFL7). MATERIALS AND METHODS: Resected formalin-fixed paraffin-embedded MPM tissues from 29 patients, 14 patient-matched non-neoplastic pleura (NNP) specimens, 5 MPM diagnostic biopsies (DB), and 5 samples of pneumothorax-induced benign reactive mesothelial proliferation (PTHX) were analyzed. miR-126 and EGFL7 mRNA were quantified by RT-qPCR. CpG-islands' methylation in the EGFL7 promoter was analyzed using methylation-specific PCR and in the MIR126-containing intron 7 was quantified by pyrosequencing. RESULTS: Relative to NNP, EGFL7 was under-expressed more than 4-fold in MPM (p<0.001). EGFL7 mRNA and miR-126 levels correlated in MPM (p<0.01) and NNP (p<0.001). The EGFL7 promoter region was hypermethylated in 69% of MPM and 80% of DB samples, but not in NNP and PTHX samples. EGFL7 promoter hypermethylation was associated with epithelioid histology (p<0.05) and reduced patient-survival (p<0.05). CONCLUSION: In MPM, DNA-hypermethylation down-regulates miR-126 and its host gene EGFL7, therefore is a poor prognostic factor, and may represent a future therapeutic target for de-methylating strategies re-establishing EGFL7 and miR-126 expression.

Characterisation of cytotoxicity and DNA damage induced by the topoisomerase II-directed bisdioxopiperazine anti-cancer agent ICRF-187 (dexrazoxane) in yeast and mammalian cells.

BACKGROUND: Bisdioxopiperazine anti-cancer agents are inhibitors of eukaryotic DNA topoisomerase II, sequestering this protein as a non-covalent protein clamp on DNA. It has been suggested that such complexes on DNA represents a novel form of DNA damage to cells. In this report, we characterise the cytotoxicity and DNA damage induced by the bisdioxopiperazine ICRF-187 by a combination of genetic and molecular approaches. In addition, the well-established topoisomerase II poison m-AMSA is used for comparison. RESULTS: By utilizing a panel of Saccharomyces cerevisiae single-gene deletion strains, homologous recombination was identified as the most important DNA repair pathway determining the sensitivity towards ICRF-187. However, sensitivity towards m-AMSA depended much more on this pathway. In contrast, disrupting the post replication repair pathway only affected sensitivity towards m-AMSA. Homologous recombination (HR) defective irs1SF chinese hamster ovary (CHO) cells showed increased sensitivity towards ICRF-187, while their sensitivity towards m-AMSA was increased even more. Furthermore, complementation of the XRCC3 deficiency in irs1SF cells fully abrogated hypersensitivity towards both drugs. DNA-PKcs deficient V3-3 CHO cells having reduced levels of non-homologous end joining (NHEJ) showed slightly increased sensitivity to both drugs. While exposure of human small cell lung cancer (SCLC) OC-NYH cells to m-AMSA strongly induced gammaH2AX, exposure to ICRF-187 resulted in much less induction, showing that ICRF-187 generates fewer DNA double strand breaks than m-AMSA. Accordingly, when yeast cells were exposed to equitoxic concentrations of ICRF-187 and m-AMSA, the expression of DNA damage-inducible genes showed higher levels of induction after exposure to m-AMSA as compared to ICRF-187. Most importantly, ICRF-187 stimulated homologous recombination in SPD8 hamster lung fibroblast cells to lower levels than m-AMSA at all cytotoxicity levels tested, showing that the mechanism of action of bisdioxopiperazines differs from that of classical topoisomerase II poisons in mammalian cells. CONCLUSION: Our results point to important differences in the mechanism of cytotoxicity induced by bisdioxopiperazines and topoisomerase II poisons, and suggest that bisdioxopiperazines kill cells by a combination of DNA break-related and DNA break-unrelated mechanisms.