bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative Histopathological Analysis of Cervical Intra‐Epithelial Neoplasia Sections: Methodological Issues
Authors: Guillaud Martial , Cox Dennis , Malpica Anais , Staerkel Gregg , Matisic Jasenka , Van Niekirk Dirk , Adler‐Storthz Karen , Poulin Neal , Follen Michele , MacAulay Calum
Published: January 2021

Objectives: As part a Program Project to evaluate emerging optical technologies for cervical neoplasia, our group is performing quantitative histopathological analysis of biopsies from 1800 patients. Several methodological issues have arisen with respect to this analysis: (1) Finding the most efficient way to compensate for staining intensity variation with out losing diagnostic information; (2) Assessing the inter‐ and intra‐observer variability of the semi‐interactive data collection; and (3) the use of non‐overlapping cells from the intermediate layer only. Methods: Non‐overlapping quantitatively stained nuclei were selected from 280 samples with histopathological characteristics of normal (199), koilocytosis (37), CIN 1 (18), CIN 2 (10) and CIN 3 (16). Linear discriminant analysis was used to assess the diagnostic information in three different feature sets to evaluate and compare staining intensity normalization methods. Selected feature values and summary scores were used to evaluate intra‐ and inter‐observer variability. Results: The features normalized by the internal subset of the imaged cells had the same discriminatory power as those normalized by the control cells and by both normalization methods seem to have additional discriminatory power over the set of features which do not require normalization. The use of the internal subset decreased the image acquisition time by ∼50% at each center, respectively. The intra‐ and inter‐observer variability was of a similar size. Good performance was obtained by measuring the intermediate layer only. Conclusion: The use of intensity normalization from a subset of the imaged non‐overlapping intermediate layer cells works as well as or better than any of the other methods tested and provides a significant timesaving. Our intra‐ and inter‐observer variability do not seem to affect the diagnostic power of the data. Although this must be tested in a larger data set, the use of intermediate layer cells only may be acceptable when using quantitative histopathology.
Bodipy‐FL‐Verapamil: A Fluorescent Probe for the Study of Multidrug Resistance Proteins
Authors: Rosati Anna , Candussio Luigi , Crivellato Enrico , Klugmann Fiora Bartoli , Giraldi Tullio , Damiani Daniela , Michelutti Angela , Decorti Giuliana
Published: January 2021

Most of the substances used as fluorescent probes to study drug transport and the effect of efflux blockers in multidrug resistant cells have many drawbacks, such as toxicity, unspecific background, accumulation in mitochondria. New fluorescent compounds, among which Bodipy‐FL‐verapamil (BV), have been therefore proposed as more useful tools. The uptake of BV has been evaluated by cytofluorimetry and fluorescence microscopy using cell lines that overexpress P‐glycoprotein (P388/ADR and LLC‐PK1/ADR) or MRP (multidrug resistance‐related protein) (PANC‐1) and clinical specimens from patients. The effect of specific inhibitors for P‐glycoprotein (verapamil and vinblastine) or MRP (MK571 and probenecid) has been also studied. BV intracellular concentrations were significantly lower in the two P‐glycoprotein overexpressing cell lines in comparison with the parental lines. In addition, verapamil and vinblastine increased the intracellular concentrations of the dye; MK571 and probenecid, two MRP inhibitors, increased BV levels in PANC‐1 cells, that express this protein. These findings were confirmed in clinical specimens from patients. Fluorescence microscopy revealed a faint fluorescence emission in P‐glycoprotein or MRP expressing cell lines; however, treatment with specific inhibitors significantly increased the fluorescence. BV is a useful tool for studying multidrug resistance proteins with different techniques such as cytofluorimetry and fluorescence microscopy, but does not discriminate between P‐glycoprotein and MRP. In comparison with other classic fluorescent probes, the assay with this dye is extremely rapid, simple, not toxic for cells, devoid of fluorescent background, and can be useful in the clinical settings.
PHOTOPROBER® Biotin: An Alternative Method for Labeling Archival DNA for Comparative Genomic Hybridization
Authors: Korinth Dirk , Donhuijsen Konrad , Bockmühl Ulrike , Petersen Iver
Published: January 2021

Comparative genomic hybridization (CGH) represents a powerful method for screening the entire genome of solid tumors for chromosomal imbalances. Particularly it enabled the molecular cytogenetic analysis of archival, formalin‐fixed, paraffin‐embedded (FFPE) tissue. A well‐known dilemma, however, is the poor DNA quality of this material with fragment sizes below 1000 bp. Nick translation, the conventionally used enzymatic DNA labeling method in CGH, leads to even shorter fragments often below a critical limit for successful analysis. In this study we report the alternative application of non‐enzymatic, PHOTOPROBE® biotin labeling for conjugation of the hapten to the DNA prior to in situ hybridization and fluorescence detection. We analyzed 51 FFPE tumor samples mainly from the upper respiratory tract by both labeling methods. In 19 cases, both approaches were successful. The comparison of hybridized metaphases showed a distinct higher fluorescence signal of the PHOTOPROBE® samples sometimes with a discrete cytoplasm background which however did not interfere with specificity and sensitivity of the detected chromosomal imbalances. For further 32 cases characterized by an average DNA fragment size below 1000 bp, PHOTOPROBE® biotin was the only successful labeling technique thus offering a new option for CGH analysis of highly degraded DNA from archival material.
Hypoxia‐Induced Acidification Causes Mitoxantrone Resistance Not Mediated by Drug Transporters in Human Breast Cancer Cells
Authors: Greijer A. E. , de Jong M. C. , Scheffer G. L. , Shvarts A. , van Diest P. J. , van der Wall E.
Published: January 2021

Hypoxia has clinically been associated with resistance to chemotherapy. The aim of this study was to investigate whether hypoxia induces resistance to doxorubicin and mitoxantrone, two common drugs in cancer treatment, in MCF‐7 breast cancer cells, and SW1573 non‐small lung cancer cells. In addition, the role of drug transporters P‐gp, BCRP and MRP1 was analysed. Hypoxia induced resistance in MCF‐7 cells to mitoxantrone shifted the IC50 value from 0.09 μM (±0.01) to 0.54 μM (±0.06) under hypoxia, whereas survival of MCF‐7 and SW1573 cells in the presence of doxorubicin was not altered. Accumulation of mitoxantrone and daunorubicin, a doxorubicin fluorescent homologue, appeared to be 5.3 and 3.2 times lower in MCF‐7 cells, respectively. Cytotoxicity assays showed no increased functionality of the drug transporters P‐gp, BCRP and MRP1 under hypoxia. In addition, protein levels of these drug transporters were not changed. Medium of the MCF‐7 cells became more acidic under hypoxia thereby causing a decreased uptake of mitoxantrone. Hypoxia induces mitoxantrone resistance in MCF‐7 cells not mediated by the three major MDR transporters. Hypoxia‐induced acidification may cause this resistance by decreased cellular uptake together with a lowered cytotoxicity due to pH‐dependent topoisomerase type II activity.
The Chromosomal Basis of Cancer
Authors: Duesberg Peter , Li Ruhong , Fabarius Alice , Hehlmann Ruediger
Published: January 2021

Conventional genetic theories have failed to explain why cancer (1) is not heritable and thus extremely rare in newborns, (2) is caused by non‐mutagenic carcinogens, (3) develops only years to decades after initiation by carcinogens, (4) follows pre‐neoplastic aneuploidy, (5) is aneuploid, (6) is chromosomally and phenotypically “unstable”, (7) carries specific aneusomies, (8) generates much more complex phenotypes than conventional mutation such as multidrug resistance, (9) generates nonselective phenotypes such as metastasis (no benefit at native site) and “immortality” (not necessary for tumorigenesis), and (10) does not contain carcinogenic mutations. We propose, instead, that cancer is a chromosomal disease. Accordingly carcinogenesis is initiated by random aneuploidies, which are induced by carcinogens or spontaneously. Since aneuploidy unbalances 1000s of genes, it corrupts teams of proteins that segregate, synthesize and repair chromosomes. Aneuploidy is therefore a steady source of chromosomal variations from which, in classical Darwinian terms, selection encourages the evolution and malignant progression of cancer cells. The rates of specific chromosomal variations can exceed conventional mutations by 4–11 orders of magnitude, depending on the degrees of aneuploidy. Based on their chromosomal constitution cancer cells are new cell “species” with specific aneusomies, but unstable karyotypes. The cancer‐specific aneusomies generate complex, malignant phenotypes through the abnormal dosages of 1000s of genes, just as trisomy 21 generates Down syndrome. In sum, cancer is caused by chromosomal disorganization, which increases karyotypic entropy. Thus, cancer is a chromosomal rather than a genetic disease. The chromosomal theory explains (1) non‐heritable cancer because aneuploidy is not heritable, (2) non‐mutagenic carcinogens as aneuploidogens, (3) long neoplastic latencies by the low probability of evolving new species, (4) nonselective phenotypes via genes hitchhiking with selective chromosomes, and (5) immortality because, through their cellular heterogeneity, cancers survive negative mutations and cytotoxic drugs via resistant subspecies.
Detection of Cervical Cancer and High Grade Neoplastic Lesions by a Combination of Liquid‐Based Sampling Preparation and DNA Measurements Using Automated Image Cytometry
Authors: Sun Xiao Rong , Wang Jian , Garner David , Palcic Branko
Published: January 2021

Objective: To establish if measurements of DNA ploidy could be used to assist cytopathologists and cytotechnologists in population based cervical cancer screening programs in countries where manually reading the slides is impossible due to the lack of sufficient skilled cytotechnologists. The goal of such program is to identify only clinically significant lesions, i.e. those where a clinical intervention to remove the lesion is required immediately. Study Design: A total of 9905 women were enrolled in the study. Cervical samples were taken with a cervix brush that was then placed into a fixative solution. The cells were separated from mucus by mechanical and chemical treatment and then deposited onto microscope slides by a cytocentrifuge. Two slides were prepared from each case; one slide was stained by Papanicolaou stain for manual cytology examination, while the other slide was stained by a DNA specific stain. The latter slide was used to determine the relative amount of DNA in the cell nuclei. Results: A total of 876 women were followed by colposcopy examination where biopsies were taken from the visible lesions or from suspicious areas and histopathology diagnosed 459 as normal or benign cases, 325 as CIN1, 36 as CIN2, 25 as CIN3/CIS, and 31 as invasive cancer. Of these 876 cases, manual cytology called 655 normal or ASCUS, 197 as LSIL, 16 cases as HSIL, and 8 as cancer. DNA measurements found 704 cases having no cells with DNA greater than 5c, 98 cases where there were 1 or 2 cells having DNA amount greater than 5c, and 74 cases where there were 3 or more cells having DNA amount greater than 5c. If manual cytology were to be used to refer all cases of HSIL and cancer to colposcopy and biopsy, 23 lesions that had to be removed would have been discovered (2 CIN2, 11 CIN3/CIS, and 10 cancers), for a sensitivity of 25.0±5.2% at specificity of 99.9±0.1%. If DNA assisted cytology were to be used instead, and all cases having 3 or more cells with DNA amount greater than 5c were to be referred to colposcopy and biopsy, then 50 lesions that had to be removed would have been discovered (10 CIN2, 15 CIN3/CIS and 25 cancers) for the sensitivity of 54.3±6.2% at specificity of 96.9±0.6%. Conclusions: The study suggests that screening for high grade cervical neoplastic lesions and cervical cancer by DNA assisted cytology could be implemented with minimal use of skilled cytotechnologists, at least in those countries where it would be difficult to introduce population based screening for cervical cancer due to the lack of availability of such skilled cytotechnologists.
The Fractal Dimension of Nuclear Chromatin as a Prognostic Factor in Acute Precursor B Lymphoblastic Leukemia
Authors: Adam Randall L. , Silva Rosana C. , Pereira Fernanda G. , Leite Neucimar J. , Lorand-Metze Irene , Metze Konradin
Published: January 2021

The fractal nature of the DNA arrangement has been postulated to be a common feature of all cell nuclei. We investigated the prognostic importance of the fractal dimension (FD) of chromatin in blasts of patients with acute precursor B lymphoblastic leukemia (B‐ALL). In 28 patients, gray scale transformed pseudo‐3D images of 100 nuclei (May–Grünwald–Giemsa stained bone marrow smears) were analyzed. FD was determined by the Minkowski–Bouligand method extended to three dimensions. Goodness‐of‐fit of FD was estimated by the R2 values in the log‐log plots. Whereas FD presented no prognostic relevance, patients with higher R2 values showed a prolonged survival. White blood cell count (WBC), age and mean fluorescence intensity of CD45 (MFICD45) were all unfavorable prognostic factors in univariate analyses. In a multivariate Cox‐regression, R2, WBC, and MFICD45, entered the final model, which showed to be stable in a bootstrap resampling study. Blasts with lower R2 values, equivalent to accentuated “coarseness” of the chromatin pattern, which may reflect profound changes of the DNA methylation, indicated a poor prognosis. In conclusion the goodness‐of‐fit of the Minkowski–Bouligand dimension of chromatin can be regarded as a new and biologically relevant prognostic factor for patients with B‐ALL.
HPV Typing and CGH Analysis for the Differentiation of Primary and Metastatic Squamous Cell Carcinomas of the Aerodigestive Tract
Authors: Will Constanze , Schewe Christiane , Schluns Karsten , Petersen Iver
Published: January 2021

Human papilloma virus (HPV) typing and Comparative Genomic Hybridisation (CGH) analysis can be used in the classification of multiple tumours of the aerodigestive tract for the differentiation between secondary malignancy versus metastasis. We present 3 exemplary cases of patients with multiple squamous cell carcinomas, localised within the head and neck region, cervical lymph node and the lung. In two patients, HPV typing identified HPV type 16 in the tonsillar carcinomas and the corresponding cervical lymph node and lung carcinoma indicating that the latter were metastatic spreads. In case 1, CGH confirmed the clonal relationship. Case two showed a peculiar syncytial growth pattern with lymphocytic infiltration which may constitute a potential morphological marker for HPV infection. In case three, a vallecular carcinoma was HPV negative while a lung cancer was positive for HPV type 6 indicating two independent primary tumours. Our case triplet illustrates the variability of HPV infection in squamous cell cancer of the aerodigestive tract and power as well as limitations of morphology, HPV typing and tumour genetics in the classification of multiple tumours.
Detection of HER2 Amplification in Breast Carcinomas: Comparison of Multiplex Ligation‐Dependent Probe Amplification (MLPA) and Fluorescence In Situ Hybridization (FISH) combined with Automated Spot Counting
Authors: Moerland Elna , van Hezik Rens L. H. P. M. , van der Aa Toine C. J. M. , van Beek Mike W. P. M. , van den Brule Adriaan J. C.
Published: January 2021

In this study the detection of HER2 gene amplification was evaluated using Fluorescence In Situ Hybridization (FISH; PathVysion) in comparison with Multiplex Ligation‐dependent Probe Amplification (MLPA), a PCR based technique. These two methods were evaluated on a series of 46 formalin fixed paraffin embedded breast carcinomas, previously tested for protein overexpression by HercepTest (grouped into Hercep 1+, 2+ and 3+). HER2 gene amplification (ratio ≥ 2.0) by FISH was found in 9/10, 10/30 and 0/6 in IHC 3+, 2+ and 1+/0 cases, respectively. Digitalized automated spot counting performed with recently developed CW4000 CytoFISH software was 100% concordant with manual FISH scoring. Using MLPA 18/46 samples showed a clear HER2 amplification. Comparing MLPA and IHC showed the same results as for FISH and IHC. All but one FISH positive cases (18/19) were confirmed by MLPA for the presence of the gene amplification. The overall concordance of detection of Her2 gene amplification by FISH and MLPA was 98% (45/46). Furthermore, both the level of amplification and equivocal results correlated well between both methods. In conclusion, MLPA is a reliable and reproducible technique and can be used as an either alternative or additional test to determine HER2 status in breast carcinomas.
Identification of the Fanconi Anemia Complementation Group I Gene, FANCI
Authors: Dorsman Josephine C. , Levitus Marieke , Rockx Davy , Rooimans Martin A. , Oostra Anneke B. , Haitjema Anneke , Bakker Sietske T. , Steltenpool Jûrgen , Schuler Dezsö , Mohan Sheila , Schindler Detlev , Arwert Fré , Pals Gerard , Mathew Christopher G. , Waisfisz Quinten , de Winter Johan P. , Joenje Hans
Published: January 2021

To identify the gene underlying Fanconi anemia (FA) complementation group I we studied informative FA‐I families by a genome‐wide linkage analysis, which resulted in 4 candidate regions together encompassing 351 genes. Candidates were selected via bioinformatics and data mining on the basis of their resemblance to other FA genes/proteins acting in the FA pathway, such as: degree of evolutionary conservation, presence of nuclear localization signals and pattern of tissue‐dependent expression. We found a candidate, KIAA1794 on chromosome 15q25‐26, to be mutated in 8 affected individuals previously assigned to complementation group I. Western blots of endogenous FANCI indicated that functionally active KIAA1794 protein is lacking in FA‐I individuals. Knock‐down of KIAA1794 expression by siRNA in HeLa cells caused excessive chromosomal breakage induced by mitomycin C, a hallmark of FA cells. Furthermore, phenotypic reversion of a patient‐derived cell line was associated with a secondary genetic alteration at the KIAA1794 locus. These data add up to two conclusions. First, KIAA1794 is a FA gene. Second, this gene is identical to FANCI, since the patient cell lines found mutated in this study included the reference cell line for group I, EUFA592.
The Carcinoma–Stromal Ratio of Colon Carcinoma Is an Independent Factor for Survival Compared to Lymph Node Status and Tumor Stage
Authors: Mesker Wilma E. , Junggeburt Jan M. C. , Szuhai Karoly , de Heer Pieter , Morreau Hans , Tanke Hans J. , Tollenaar Rob A. E. M.
Published: January 2021

Background: Tumor staging insufficiently discriminates between colon cancer patients with poor and better prognosis. We have evaluated, for the primary tumor, if the carcinoma‐percentage (CP), as a derivative from the carcinoma‐stromal ratio, can be applied as a candidate marker to identify patients for adjuvant therapy. Methods: In a retrospective study of 63 patients with colon cancer (stage I–III, 1990–2001) the carcinoma‐percentage of the primary tumor was estimated on routine H&E stained histological sections. Additionally these findings were validated in a second independent study of 59 patients (stage I–III, 1980–1992). (None of the patients had received preoperative chemo‐ or radiation therapy nor adjuvant chemotherapy.) Results: Of 122 analyzed patients 33 (27.0%) had a low CP and 89 (73.0%) a high CP. The analysis of mean survival revealed: overall‐survival (OS) 2.13 years, disease‐free‐ survival (DFS) 1.51 years for CP‐low and OS 7.36 years, DFS 6.89 years for CP‐high. Five‐year survival rates for CP‐low versus CP‐high were respectively for OS: 15.2% and 73.0% and for DFS: 12.1% and 67.4%. High levels of significance were found (OS p < 0.0001, DFS p < 0.0001) with hazard ratio’s of 3.73 and 4.18. In a multivariate Cox regression analysis, CP remained an independent variable when adjusted for either stage or for tumor status and lymph‐node status (OSp < 0.001, OSp < 0.001). Conclusions: The carcinoma‐percentage in primary colon cancer is a factor to discriminate between patients with a poor and a better outcome of disease. This parameter is already available upon routine histological investigation and can, in addition to the TNM classification, be a candidate marker to further stratify into more individual risk groups.
Cell‐cycle and Apoptosis Regulators (p16INK4A, p21CIP1, α‐Catenin, Survivin, and hTERT) and Morphometry‐Defined MPECs Predict Metachronous Cancer Development in Colorectal Adenoma Patients
Authors: Soreide Kjetil , Buter Tirza C. E. , Janssen Emiel A. , Gudlaugsson Einar , Skaland Ivar , Körner Hartwig , Baak Jan P. A.
Published: January 2021

Background and Aims: Although adenomas may be precursors to colorectal cancers (CRC), knowledge concerning the development of metachronous CRC is scarce. We assessed whether differential expression of cell‐cycle and apoptosis‐regulating proteins and a monotonous population of elongated cells (MPECs) in colorectal adenomas could predict metachronous CRC. Methods: Application of immunohistochemistry on tissue microarrays in consecutive, population‐based colorectal adenomas. Influence of classic features (e.g., intraepithelial neoplasia grade, histological type, size) was examined. Results: Of 171 patients with colorectal adenoma 86% (n = 147) were eligible for study; 10 (7%) developed metachronous CRC. Median time to cancer was 69 months (range, 25–256). Median follow‐up was equal for the non‐cancer and cancer groups. Elevated expression of cellcycle regulators p16INK4A, p21CIP1, and cytoplasmic/nuclear α‐catenin correlated with increased CRC risk (all P <0.0001), as did elevated expression of the anti‐apoptosis protein survivin (P <0.0001) and human telomerase reverse transcriptase (hTERT; P <0.001). Survivin, hTERT, and nuclear α‐catenin were the most predictive molecular markers (hazard ratios [HRs]: 6.3, 9.4, and 5.8, respectively). In a combined multivariate model, MPECs had the best overall prognostic ability (HR 28.2, 95% CI: 3.6–223.0), together with survivin, and hTERT. Within adenomas containing MPECs, several molecular markers further defined high‐risk patients. Conclusions: Among several markers predictive for metachronous CRC development in colorectal adenomas, MPECs, survivin and hTERT may, when validated, provide information superior to conventional histology, with relevance for the clinical management of patients with colorectal adenoma.
CD31 and CD34 Expression as Immunohistochemical Markers of Endothelial Transdifferentiation in Human Cutaneous Melanoma
Authors: Pisacane A. M. , Picciotto F. , Risio M.
Published: January 2021

Introduction: Vasculogenic mimicry, as previously described in aggressive melanoma, is characterized by the de novo generation of intratumoral patterned vascular channels, composed of PAS‐positive basement membrane in the absence of endothelial cells, providing additional microcirculation, in support to the classic tumoral angiogenesis. Methods: We investigated the immunohistochemical expression of two endothelial markers, CD31 and CD34, in tumoral cells of 60 melanomas (45 primary cutaneous and 15 metastatic) as possible evidence of vasculogenic mimicry. In addition we investigated the relationship between CD31 and CD34 expression and three pathological markers such as Clark’s level, and skin ulceration, predictive of melanoma’s aggressive behaviour, and mitotic index. Results: No cases of common melanocytic nevi immunoreacted with CD31 or CD34. Random CD31 immunoreactivity was present in 6% of Clark’s level I/II, 50% of Clark′s level III and 80% Clark′s level IV/V. CD34 was negative in Clark′s level I/II but randomly stained the 20% and 55% of level III and IV/V respectively. 66% (10/15) of metastatic melanomas were CD31 positive showing a canalicular immunostaining pattern, conversely CD34 expression was never found. 7/8 cutaneous ulcerated melanomas immunostained for CD31 and 4/8 for CD34. CD31 immunostained 88% high/intermediate MI, and 53% of low MI melanomas. CD34 decorated the 29% of high/intermediate and 38% of low MI melanomas. Conclusions: CD31 and CD34 immunoreactivity closely parallel both the different morphologic steps of melanocytic tumor progression and the presence of histological parameters related to the aggressive behaviour. Their expression could be related to endothelial transdifferentiation of melanoma cells although a consequent functional role has not been demonstrated yet.
Independent Induction of Caspase‐8 and cFLIP Expression during Colorectal Carcinogenesis in Sporadic and HNPCC Adenomas and Carcinomas
Authors: Heijink D. M. , Kleibeuker J. H. , Jalving M. , Boersma-van Ekb W. , Koornstra J. J. , Wesseling J. , de Jong S.
Published: January 2021

Background: TNF‐Related Apoptosis Inducing Ligand (TRAIL) is a promising agent for the induction of apoptosis in neoplastic tissues. Important determinants of TRAIL sensitivity are two intracellular proteins of the TRAIL pathway, caspase‐8 and its anti‐apoptotic competitor cellular Flice‐Like Inhibitory Protein (cFLIP). Methods: The aim of this study was to investigate basic expression of caspase‐8 and cFLIP in normal colorectal epithelium (n = 20), colorectal adenomas (n = 66) and colorectal carcinomas (n = 44) using immunohistochemistry performed on both sporadic and Hereditary Non‐Polyposis Colorectal Cancer (HNPCC or Lynch syndrome)‐associated adenomas and carcinomas. Results: Expression of both caspase‐8 and cFLIP was similar in cases with sporadic and hereditary origin. Expression of caspase‐8 in colorectal adenomas and carcinomas was increased when compared to normal colon tissue (P = 0.02). Nuclear, paranuclear as well as cytoplasmic localizations of caspase‐8 were detected. Immunohistochemistry revealed an upregulation of cFLIP in colorectal carcinomas in comparison to normal epithelium and colorectal adenomas (P < 0.001). A large variation in the caspase‐8/cFLIP ratio was observed between the individual adenomas and carcinomas. Conclusion: Caspase‐8 and cFLIP are upregulated during colorectal carcinogenesis. Upregulation of caspase‐8 and/or downregulation of cFLIP may be interesting approaches to maximize TRAIL sensitivity in colorectal neoplasms.
Pathology Databanking and Biobanking in The Netherlands, a Central Role for PALGA, the Nationwide Histopathology and Cytopathology Data Network and Archive
Authors: Casparie M. , Tiebosch A. T. M. G. , Burger G. , Blauwgeers H. , van de Pol A. , van Krieken J. H. J. M. , Meijer G. A.
Published: January 2021

Since 1991, a nationwide histopathology and cytopathology network and archive is in operation in The Netherlands under the name PALGA, encompassing all sixty‐four pathology laboratories in The Netherlands. The overall system comprises decentralized systems at the participating laboratories, a central databank, and a dedicated communication and information exchange tool. Excerpts of all histopathology and cytopathology reports are generated automatically at the participating laboratories and transferred to the central databank. Both the decentralized systems and the central system perform checks on the quality and completeness of excerpts. Currently, about 42 million records on almost 10 million patients are stored in the central databank. Each excerpt contains patient identifiers, including demographic data and the so‐called PALGA diagnosis. The latter is structured along five classification axes: topography, morphology, function, procedure, and diseases. All data transfer and communication occurs electronically with encryption of patient and laboratory identifiers. All excerpts are continuously available to all participating pathology laboratories, thus contributing to the quality of daily patient care. In addition, external parties may obtain permission to use data from the PALGA system, either on an ongoing basis or on the basis of a specific permission. Annually, 40 to 60 applications for permission to use PALGA data are submitted. Among external users are the Dutch cancer registry, population‐based screening programs for cancer of the uterine cervix and breast cancer in The Netherlands, and individual investigators addressing a range of research questions. Many scientific papers and theses incorporating PALGA data have been published already. In conclusion, the PALGA system is a unique system that requires a minimal effort on the part of the participating laboratories, while providing them a powerful tool in their daily practices.
A comparative Analysis by SAGE of Gene Expression Profiles of Esophageal Adenocarcinoma and Esophageal Squamous Cell Carcinoma
Authors: van Baal Jantine W. P. M. , Milana Francesco , Rygiel Agnieszka M. , Sondermeijer Carine M. T. , Spek C. Arnold , Bergman Jacques J. G. H. M. , Peppelenbosch Maikel P. , Krishnadath Kausilia K.
Published: January 2021

Esophageal adenocarcinoma (EA) and esophageal squamous cell carcinoma (ESCC) are the two main types of esophageal cancer. Despite extensive research the exact molecular basis of these cancers is unclear. Therefore we evaluated the transcriptome of EA in comparison to non‐dysplastic Barrett’s esophagus (BE), the metaplastic epithelium that predisposes for EA, and compared the transcriptome of ESCC to normal esophageal squamous epithelium. For obtaining the transcriptomes tissue biopsies were used and serial analysis of gene expression (SAGE) was applied. Validation of results by RT‐PCR and immunoblotting was performed using tissues of an additional 23 EA and ESCC patients. Over 58,000 tags were sequenced. Between EA and BE 1013, and between ESCC and normal squamous epithelium 1235 tags were significantly differentially expressed (p < 0.05). The most up‐regulated genes in EA compared to BE were SRY‐box 4 and Lipocalin2, whereas the most down‐regulated genes in EA were Trefoil factors and Annexin A10. The most up‐regulated genes in ESCC compared to normal squamous epithelium were BMP4, E‐Cadherin and TFF3. The results could suggest that the BE expression profile is closer related to normal squamous esophagus then to EA. In addition, several uniquely expressed genes are identified.
Conditional Inactivation of HIF-1 Using Intrabodies
Authors: Groot Arjan J. , Gort Eelke H. , van der Wall Elsken , van Diest Paul J. , Vooijs Marc
Published: January 2021

Hypoxia is a hallmark of solid cancers and triggers the transcription of genes responsible for cell survival. The transcription factor Hypoxia-Inducible Factor 1 (HIF-1) is a key regulator in this response and frequently activated in human cancer. HIF-1 activation is associated with tumor aggressiveness and poor clinical outcome and, therefore, may provide an attractive therapeutic target. Here we provide a novel approach for HIF-1 targeted therapy using single-domain llama antibodies directed against the HIF-1α oxygen dependent degradation domain which encompass the N-terminal transactivation domain. Conditional expression of HIF intrabodies in mammalian cells interfered with binding to pVHL and inhibited hypoxia induced activation of endogenous target genes. Inducible intrabody targeting is a highly specific strategy for temporal protein inactivation and may have applications for disease treatment.
Mitochondrial DNA Depletion Reduces PARP-1 Levels and Promotes Progression of the Neoplastic Phenotype in Prostate Carcinoma
Authors: Moro Loredana , Arbini Arnaldo A. , Marra Ersilia , Greco Margherita
Published: January 2021

Mitochondrial dysfunction resulting from mitochondrial DNA (mtDNA) mutations and/or depletion has been correlated with cancer progression and drug resistance. To investigate the role of mtDNA in prostate cancer progression, we used LNCaP and PC-3 prostate carcinoma cells as experimental model. Compared to minimally invasive androgen-dependent LNCaP cells, highly invasive androgen-independent PC-3 cells, as well as androgen-independent DU145 and C4-2 cells, exhibited significantly reduced mtDNA content. In PC-3 cells, reduction of mtDNA was accompanied by decreased mitochondrial membrane potential (ΔΨm), increased migration onto the basement membrane protein laminin-1, reduced chemosensitivity to paclitaxel (IC50=110 nM vs. 22 nM) and decreased expression of poly(ADP-ribose) polymerase (PARP)-1. To investigate the relationship between mtDNA depletion and these phenotypic characteristics, we established mtDNA-depleted LNCaP cells [Rho(−)] by long-term exposure to ethidium bromide or treated wild-type LNCaP cells with a mitochondrial ionophore, carbonyl cyanide m-chlorophenylhydrazone. Both manipulations resulted in ΔΨm loss, acquisition of invasive cytology, increased motility onto laminin-1, reduced sensitivity to paclitaxel (IC50=~100 nM) and ~75% reduction in PARP-1 protein levels, resembling PC-3 cells. Overall, these results provide novel evidence demonstrating that mtDNA depletion in early prostate carcinoma may contribute to the acquisition of a more invasive phenotype that is less sensitive to paclitaxel-induced apoptosis.
Relationship between Dyskerin Expression and Telomerase Activity in Human Breast Cancer
Authors: Montanaro Lorenzo , Calienni Maria , Ceccarelli Claudio , Santini Donatella , Taffurelli Mario , Pileri Stefano , Treré Davide , Derenzini Massimo
Published: January 2021

The nucleolar protein dyskerin is involved in the modification of specific uridine residues to pseudouridine on ribosomal and small nuclear RNAs and in the stabilization of the telomerase RNA component (TERC). In this study we investigated for the first time the relationship between dyskerin expression and telomerase activity in a series of 61 primary breast carcinomas. We found that when dyskerin mRNA values were very low the telomerase activity was markedly reduced, independently of the expression of other important components of the telomerase complex such as telomerase reverse transcriptase (TERT). In vitro experiments showed that reduction of dyskerin expression affect telomerase activity through the reduction of TERC. Only when TERC levels were strongly reduced telomerase activity was hindered. Retroviral mediated over-expression of TERC abolished the telomerase impairment due to dyskerin knock down. In conclusion, our results indicated that, beside its effect on ribosome biogenesis, the levels of dyskerin in cancer cells modulate telomerase activity through the regulation of TERC levels, independently of TERT expression. This should be taken into consideration when utilizing TERT expression as a surrogate indicator of telomerase activity in tumour pathology.
HCC Heterogeneity: Molecular Pathogenesis and Clinical Implications
Authors: Fransvea Emilia , Paradiso Angelo , Antonaci Salvatore , Giannelli Gianluigi
Published: January 2021

Background: Hepatocellular carcinoma (HCC) poses a major challenge because of the extreme variability of the clinical outcome, which makes it difficult to properly stage the disease and thereby estimate the prognosis. There is growing evidence that this heterogeneous clinical behavior is attributable to several different biological pathways. A novel approach to mapping these differences is by investigating the epigenetics associated with certain clinical aspects.

Design: Herein, the relevance of these molecular differences in combination with the biological and molecular pathways regulating the clinical outcome will be discussed. Use of a mechanistic and pathogenic approach to clarify the natural history of HCC is not just an academic speculation but should help to develop new therapies and to tailor these therapies to each individual patient.

Conclusion: New biological therapies targeting components of the tumoral or peritumoral microenvironment are crucial to the fight against HCC. However, biological redundancies and the presence of several growth factors, hormones, cytokines, etc., potentially involved in HCC tumor progression make it difficult to assess the best target. Sorafenib, a multi-tyrosine kinase inhibitor, blocks the functions of different growth factors present in the tissue microenvironment. The use of Sorafenib in patients with HCC offers a new approach to the therapy of this disease, stimulating research focusing on the development of drugs based on new molecular and pathogenic insights.
Comparison between Real-Time Quantitative PCR Detection of HER2 mRNA Copy Number in Peripheral Blood and ELISA of Serum HER2 Protein for Determining HER2 Status in Breast Cancer Patients
Authors: Savino Maria , Parrella Paola , Copetti Massimiliano , Barbano Raffaela , Murgo Roberto , Fazio Vito Michele , Valori Vanna Maria , Carella Massimo , Garrubba Maria , Santini Stefano Angelo
Published: January 2021

Background: The development of non-invasive procedure to determine HER2 status may represent a powerful method for monitoring disease progression and response to the treatment.

Methods: Serum samples and RNA from peripheral blood were evaluated in 85 breast cancer patients (49 HER2 positive and 36 HER2 negative) and 22 healthy controls. HER2 mRNA levels were measured by real-time quantitative PCR (QPCR) and serum HER2 protein by immunoenzimatic assay (EIA). ROC curve analyses were used to determine the optimal cut off values.

Results: A statistically significant difference was detected for both QPCR and EIA in HER2 positive patients as compared with both healthy controls and HER2 negative tumours. QPCR showed a 91% (CI95%: 84%–98%) specificity and a 78% (CI95%: 68%–88%) sensitivity for an optimal cut off value of 4.74. The optimal cut off value for EIA was 22 ng/ml yielding a 95% (CI95%: 90%–100%) specificity and a 59% (CI95%: 48%–70%) sensitivity. The QPCR assay was slightly less specific than EIA in discriminating HER2 positive breast cancers from HER2 negative tumours (78% CI95%: 69%–87% versus 86% CI95%: 79%–93%), but it was more sensitive (76% CI95%: 67%–85% versus 55% CI95%: 44%–66%).

Conclusions: Our results indicate that QPCR performs better than EIA in the determination of HER2 status of breast cancer patients and could be useful in monitoring the disease during follow up.
Pla2g2a Attenuates Colon Tumorigenesis in Azoxymethane-Treated C57BL/6 Mice; Expression Studies Reveal Pla2g2a Target Genes and Pathways
Authors: Fijneman Remond J. A. , Bade Lindsey K. , Peham Johannes R. , van de Wiel Mark A. , van Hinsbergh Victor W. M. , Meijer Gerrit A. , O’Sullivan Michael G. , Cormier Robert T.
Published: January 2021

Background: The group IIA secretory phospholipase A2 gene, Pla2g2a, confers resistance to intestinal tumorigenesis in the ApcMin/+ mouse model. However, it is unclear how Pla2g2a exerts its tumor-suppressive effects and whether its mode of action depends on Apc-germline mutations.

Methods: We tested whether expression of a Pla2g2a transgene provides protection against carcinogen-induced colon tumors, and examined whether the normal colon microenvironment is modulated by Pla2g2a expression.

Results: Pla2g2a strongly inhibited colon tumorigenesis in mice following treatment with the DNA alkylating agent azoxymethane (AOM). Moreover, AOM-induced duodenal tumors were also attenuated by Pla2g2a expression. These tumors demonstrated upregulation of β-catenin, indicative of involvement of the Wnt signaling pathway. Comparison of genome-wide microarray expression profiles of healthy (non-pathologic) colon tissues from Pla2g2a-transgenic to non-transgenic mice revealed 382 genes that were differentially expressed, comprising clusters of genes involved in inflammation and microbial defense, cell signaling and cell cycle, transactivation, apoptosis and mitochondrial function, DNA repair, and lipid and energy metabolism. Pathway analysis using Gene Set Enrichment Analysis (GSEA) indicated that Pla2g2a suppresses the expression of interferon-induced genes.

Conclusion: Our results demonstrate that Pla2g2a attenuates colon tumorigenesis independent of Apc-germline mutations, and reveal Pla2g2a target genes and pathways in non-pathologic colon microenvironment that influence conditions for colorectal cancer development.
Multiple Localization of Endogenous MARK4L Protein in Human Glioma
Authors: Magnani Ivana , Novielli Chiara , Bellini Melissa , Roversi Gaia , Bello Lorenzo , Larizza Lidia
Published: January 2021

Background: We have previously shown that the sustained expression of MARK4L transcripts in glioma and neural progenitors (NHNPs) declines after exposure to antisense MARK4L oligonucleotides in glioblastoma cell lines. Array-CGH confirmed the genomic duplication of MARK4L identified by FISH in a glioblastoma cell line. This background together with literature data on the exogenous association of MARK4 with interphase centrosome prompted us to investigate the sub-cellular localization of the endogenous MARK4L protein aiming at achieving insights on its possible role in the pathomechanisms of glioma.

Methods: Immunodetection was carried out to validate the specificity of MARK4L antibody in gliomas and NHNPs. Mass spectrometry was applied for MARK4L protein identification in a representative glioblastoma cell line. Combined biochemical fractionation and immunodetection analyses were performed to confirm the sub-cellular localization of MARK4L achieved by immunofluorescence in glioma cell lines.

Results: By assigning MARK4L protein within the band immunoprecipitated by the specific antibody we validated our anti-MARK4L antibody. We demonstrated that the endogenous MARK4L: (i) colocalizes with centrosomes at all mitotic stages and resides in centrosome-enriched fractions; (ii) associates with the nucleolus and the midbody and respective fractions, and (iii) co-stains the aberrant centrosome configurations observed in glioma cell lines.

Conclusions: The overall data merge on the multiplex entry of MARK4L into the cell cycle and link it to the aberrant centrosomes in glioma cell lines suggesting a possible role of this kinase in the abnormal mitotic processes of human glioma.
A Panel of High Resolution Melting (HRM) Technology-Based Assays with Direct Sequencing Possibility for Effective Mutation Screening of EGFR and K-ras Genes
Authors: Heideman D. A. M. , Thunnissen F. B. , Doeleman M. , Kramer D. , Verheul H. M. , Smit E. F. , Postmus P. E. , Meijer C. J. L. M. , Meijer G.A. , Snijders P. J. F.
Published: January 2021

Background: Increasing data from clinical trials support EGFR and K-ras mutation status as predictive markers of tumour response to EGFR-targeted therapies. Consequently, rapid and reliable mutation screening assays are demanded to guide rational use of EGFR-targeted therapies.

Methods: In this study, we describe the development of high resolution melting (HRM) technology-based assays with direct sequencing confirmation possibility for mutation screening of the EGFR gene (exons 19, 20 and 21) in routine diagnostic specimens, and compared assay findings to those of conventional nested-PCR following cycle-sequencing.

Results: In reconstruction experiments, each HRM assay following sequencing demonstrated a sensitivity of ≤5% of mutated DNA in a background of wild-type DNA. The panel of EGFR HRM assays following sequencing applied to a series of genomic DNA samples isolated from 68 FFPE NSCLC specimens correctly identified all EGFR mutations that were previously found by nested-PCR following cycle-sequencing. The HRM approach additionally scored two mutations not detected by the conventional assay.

Complementary HRM following sequencing for K-ras revealed three mutations. EGFR and K-ras mutations were mutually exclusive.

Conclusions: The panel of designed HRM assays with direct reflex sequencing possibility provides an effective method for mutation screening of EGFR and K-ras genes in routine diagnostic specimens, thereby allowing the selection of the treatment of choice in clinical practice.
TGF-β Repressors SnoN and Ski Are Implicated in Human Colorectal Carcinogenesis
Authors: Bravou Vasiliki , Antonacopoulou Anna , Papadaki Helen , Floratou Konstantina , Stavropoulos Michalis , Episkopou Vasso , Petropoulou Chariklia , Kalofonos Haralabos
Published: January 2021

Background: The TGF-β signaling repressors SnoN and Ski have been critically implicated in human cancer.

Methods: To explore the role of SnoN and Ski in the development and progression of colorectal cancer we examined their protein expression profile by immunohistochemistry in a series of human colorectal adenomas, carcinomas and lymph node metastases. The mRNA expression of SnoN was also quantified by Real-Time RT-PCR.

Results: SnoN and Ski were overexpressed both in adenomas with severe dysplasia and colorectal carcinomas. Protein expression was cytoplasmic and nuclear with predominant cytoplasmic localization. The subcellular localization was related differently to pathologic variables of colorectal carcinomas. Although there was no significant association of protein levels with tumor invasion and metastasis, a significant correlation of nuclear SnoN and Ski with β-catenin pathway was observed. Moreover, SnoN mRNA did not differ in carcinomas as compared to normal control and there was no correlation between SnoN protein and mRNA levels.

Conclusion: Our findings suggest that SnoN and Ski exert oncogenic effects in human colorectal carcinogenesis and their overexpression is implicated in early stage disease.
Different Subcellular Localization of ALCAM Molecules in Neuroblastoma: Association with Relapse
Authors: Corrias Maria Valeria , Gambini Claudio , Gregorio Andrea , Croce Michela , Barisione Gaia , Cossu Claudia , Rossello Armando , Ferrini Silvano , Fabbi Marina
Published: January 2021

Background: The Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD), involved in nervous system development, has been linked to tumor progression and metastasis in several tumors. No information is available on ALCAM expression in neuroblastoma, a childhood neoplasia originating from the sympathetic nervous system.

Methods: ALCAM expression was analysed by immunofluorescence and immunohistochemistry on differentiated neuroblastoma cell lines and on archival specimens of stroma-poor, not MYCN amplified, resectable neuroblastoma tumors, respectively.

Results: ALCAM is variously expressed in neuroblastoma cell lines, is shed by metalloproteases and is cleaved by ADAM17/TACE in vitro. ALCAM is expressed in neuroblastoma primary tumors with diverse patterns of subcellular localization and is highly expressed in the neuropil area in a subgroup of cases. Tumor specimens showing high expression of ALCAM at the membrane of the neuroblast body or low levels in the neuropil area are associated with relapse (P = 0.044 and P < 0.0001, respectively). In vitro differentiated neuroblastoma cells show strong ALCAM expression on neurites, suggesting that ALCAM expression in the neuropil is related to a differentiated phenotype.

Conclusions: Assessment of ALCAM localization by immunohistochemistry may help to identify patients who, in the absence of negative prognostic factors, are at risk of relapse and require a more careful follow-up.