bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Predisposition to Sporadic Cancer: How to Handle Major Effects of Minor Genes?
Authors: Fijneman Remond J. A.
Published: January 2021

Predisposition to non‐familial, sporadic cancer is strongly influenced by multiple tumor susceptibility genes (TSGs), each with apparently minor effects on the cancer phenotype. Sequence analysis of the human genome has yielded numerous single nucleotide polymorphisms (SNPs), raising the expectation that new low‐penetrance TSGs will be identified that can be used to estimate an individuals cancer risk. However, mouse models for human cancer showed that the effects of many low‐penetrance TSGs are highly variable due to their involvement in epistatic interactions. Together, these interacting TSGs form large molecular networks, which represent cancer‐associated biological modules that influence the tumorigenic process. As a consequence, although allelic variation in one TSG on a permissive genetic Background can have major effects on tumor development, the net effect of allelic variation in multiple interacting TSGs remains hard to predict. Therefore, the predictive value of SNP‐analysis to estimate an individuals cancer risk will be restricted to those TSGs that exhibit single‐gene effects. New strategies need to be developed to evaluate cancer risk associated with biological modules that are influenced by TSG‐networks.
Phenotypic Changes in Mitochondrial Membrane Potential (Δψm) during Valinomycin‐Induced Depolarisation and Apoptosis
Authors: Morrison M. L. , Williamson K. , Arthur K. , Price G. J. , Hamilton P. W. , Maxwell P.
Published: January 2021

A large body of evidence has implicated mitochondria in control of cell death, where key apoptotic mechanisms involve change in mitochondrial membrane permeability and depolarisation of mitochondrial membrane potential (Δψm). Assessment of Δψm is traditionally conducted using the lipophilic cation JC‐1 on the flow cytometer or by fluorescent microscopy. Here we assess JC‐1 aggregation using the novel tool of digital texture analysis to establish mitochondrial phenotypic changes induced by the K+ ionophore, valinomycin in a unique model comprising SW480 and SW620 cell lines. This provides an opportunity to study these phenomena in the context of colorectal cancer. Valinomycin‐induced apoptosis was detected using morphology and analysis of DNA content. Cells were treated with valinomycin, images digitally recorded on a calibrated video photometer and subjected to high resolution digital texture analysis. This demonstrated that the HARAM texture features (Mean of the Haralick texture features) were highly valuable in describing the transition of Δψm as the cell undergoes apoptosis. In Conclusion this study illustrates the potential of texture analysis as a novel and additional technique for quantifying JC‐1 aggregation and revealing the spectrum of collapse of Δψm during apoptosis.
Molecular Cytogenetics: Genomic Imbalances in Colorectal Cancer and their Clinical Impact
Authors: Grade Marian , Becker Heinz , Liersch Torsten , Ried Thomas , Ghadimi B. Michael
Published: January 2021

Chromosomal aberrations play a dominant role in colorectal carcinogenesis. The application of fluorescence in situ hybridization (FISH) based techniques such as comparative genomic hybridization (CGH) and spectral karyotyping (SKY) revealed that colorectal carcinomas are characterized by a specific pattern of chromosomal imbalances which sequentially accumulate during cancer progression. This review aims to summarize molecular cytogenetic studies, provides a background on the functional relevance of chromosomal aberrations for colorectal cancer progression and discusses their potential clinical impact.
Survivin is Highly Expressed and Promotes Cell Survival in Malignant Peritoneal Mesothelioma
Authors: Zaffaroni Nadia , Costa Aurora , Pennati Marzia , De Marco Cinzia , Affini Emanuela , Madeo Maria , Erdas Roberta , Cabras Antonello , Kusamura Shigeki , Baratti Dario , Deraco Marcello , Daidone Maria Grazia
Published: January 2021

Background: The biology of malignant peritoneal mesothelioma (MPM) is largely unknown. In the present study, we assessed the expression of survivin and other members of the inhibitors of apoptosis proteins (IAP) family (IAP‐1, IAP‐2 and X‐IAP) in a series of 32 MPM surgical specimens and investigated the effects of survivin knockdown in an established MPM cell line. Methods: Expression of different IAPs was measured by immunohistochemistry. MPM cells were transfected with a small‐interfering RNA (siRNA) targeting survivin mRNA and analyzed for survivin expression, growth rate, and ability to undergo spontaneous and drug (cisplatin, doxorubicin)‐induced apoptosis. Results: Survivin expression was observed in 29 (91%) surgical MPM specimens, whereas the positivity rate for the other IAPs ranged from 69% to 100%. Transfection of MPM cells with the survivin siRNA induced a marked inhibition of survivin protein expression, a time‐dependent decline in cell growth and an enhanced rate of spontaneous and drug‐induced apoptosis, with a concomitant increase in the catalytic activity of caspase‐9. Conclusion: Our results show for the first time that survivin, as well as other IAPs, is largely expressed in clinical MPMs and suggest that strategies aimed at down‐regulating survivin may provide a novel approach for the treatment of the malignancy.
Objective Assessment of Cancer Biomarkers Using Semi‐Rare Event Detection
Authors: van der Laak Jeroen A. W. M. , Siebers Albertus G. , Aalders Sabine A. A. P. , Grefte Johanna M. M. , de Wilde Peter C. M. , Bulten Johan
Published: January 2021

Objective and reproducible assessment of cancer biomarkers may be performed using rare event detection systems. Because many biomarkers are not true ‘rare events’, in this study a semi‐rare event detection system was developed. The system is capable of assigning a discriminant score to detected positive cells, expressing the extent and intensity of the immunocytochemical staining. A gallery image is constructed showing the diagnostically most interesting cells as well as quantitative data expressing the biomarker staining pattern. To increase scanning speed, an adaptive scanning strategy is studied in which scanning is aborted when a sufficient number of positive cells has been identified. System performance was evaluated using liquid based cervical smears, stained with an antibody directed against p16INK4a tumor suppressor protein. Overexpression of p16INK4a in cervix is related to high‐risk HPV infection, which is associated with carcinogenesis. Reproducibility of the system was tested on specimens containing limited positivity. Quantitative analysis was evaluated using 10 cases within normal limits and 10 high grade lesions. The system was highly reproducible in detecting positive cells and in calculating discriminant scores (average CV 0.7%). Quantitative features were significantly increased in high grade lesions (p < 0.001). Adaptive scanning decreased scanning time with only minor impact on scanning results. The system is capable of automated, objective and reproducible assessment of biomarker expression and may be useful for a variety of applications.
BRAF in Papillary Thyroid Carcinoma
Authors: Lanzilotta Salvatore Giovanni , Grammatica Luciano , Paradiso Angelo , Simone Gianni
Published: January 2021

Novel genetic findings about papillary thyroid carcinoma identify BRAF gene as a subject of great interest. Involvement of BRAF gene in pathogenesis of PTC, diagnostic value and the putative prognostic significance of its T1799A mutation are summarized in this article. Furthermore, a particular attention is focused to the role of pre‐operative detection of BRAF mutation in the FNAB specimens of thyroid nodules and to the use of this gene as target for an effective cancer therapy.
655Val and 1170Pro ERBB2 SNPs in Familial Breast Cancer Risk and BRCA1 Alterations
Authors: Tommasi Stefania , Fedele Vita , Lacalamita Rosanna , Bruno Michele , Schittulli Francesco , Ginzinger David , Scott Gery , Eppenberger-Castori Serenella , Calistri Daniele , Casadei Silvia , Seymour Ian , Longo Salvatore , Giannelli Gianluigi , Pilato Brunella , Simone Giovanni , Benz Christopher C. , Paradiso Angelo
Published: January 2021

Human ERBB2 presents several SNPs. One of these, Ile655Val, introduces a structural change in the transmembrane region of ERBB2 and has been the focus of debate over its potential role as a susceptibility marker for breast cancer risk. Another SNP, Ala1170Pro, introduces a structural change in the carboxyl‐terminal regulatory domain of the protein, but its clinical and biological importance remains undefined. The aim of this study was to investigate the association of rare alleles of both SNPs and the risk of developing breast cancer, BRCA1 alterations and clinical‐pathological features of Caucasian breast cancer patients with familial history of breast/ovarian cancer. The originality of the present paper is that it is the only specifically focusing on the relationship between ERBB2 SNPs and familiarity/BRCA1 characteristics. A consecutive series of 628 patients with first diagnosis of breast cancer and 169 healthy people had DNA analyzed for both SNPs. Genotypic or allelic frequencies of ERBB2 SNPs in breast cancer patients were similar than in controls. The variant allele 655Val was significantly associated with younger age (p=0.009) particularly associated with patient family history of breast cancer (p=0.02). The 655Val allele was also more commonly found in invasive, while the variant 1170Pro in estrogen receptor positive breast cancers. Furthermore, this last SNP seems to be strictly associated with the presence of BRCA1 polymorphisms. In conclusion, these findings point to the existence of an association of ERBB2 allelic variants at both loci with specific breast tumor phenotypes and to the need of deeply investigate different gene SNPs association for risk defining.
Short Interfering RNA Directed against the SLUG Gene Increases Cell Death Induction in Human Melanoma Cell Lines Exposed to Cisplatin and Fotemustine
Authors: Vannini Ivan , Bonafe Massimiliano , Tesei Anna , Rosetti Marco , Fabbri Francesco , Storci Gianluca , Ulivi Paola , Brigliadori Giovanni , Amadori Dino , Zoli Wainer
Published: January 2021

Background: Melanoma remains largely resistant to currently available chemotherapy, and new strategies have been proposed to flank standardized therapeutic protocols in an effort to improve efficacy. Such an approach requires good knowledge of the mechanisms involved in the resistance and survival of melanoma cells. In this context, the SLUG gene has recently been characterized as a major regulator of melanocytes and melanoma cell survival. Methods: We tested the hypothesis that an oligonucleotide‐based short interfering RNA (siRNA) directed against the SLUG gene increases the susceptibility of melanoma cells to drugs such as cisplatin and fotemustine, which are frequently used to treat this cancer. Results: It was found that SLUG siRNA increased cisplatin‐induced cell death and rendered the drug active in vitro at half its plasmatic peak concentration. Such activity was correlated with an upregulation of the pro‐apoptotic gene, PUMA. Furthermore, SLUG siRNA increased the capacity of fotemustine to elicit cell death and induced p21WAF1 upregulation, resulting in cell cycle arrest. Interestingly, this pathway did not require functional p53. Conclusion: These findings suggest that SLUG siRNA enhances the efficacy of two of the most widely used drugs to treat melanoma.
Comparing the Prognostic Value of PTEN and Akt Expression with the Mitotic Activity Index in Adjuvant Chemotherapy‐Treated Node‐Negative Breast Cancer patients aged <55 years
Authors: Janssen Emiel A. M. , Søiland Håvard , Skaland Ivar , Gudlaugson Einar , Kjellevold Kjell H. , Nysted Arne , Søreide Jon-Arne , Baak Jan P. A.
Published: January 2021

Background: The prognostic value of the PI3K/Akt/mTOR pathway and PTEN in invasive breast cancer (IBC) is controversial. Cell proliferation, especially the Mitotic Activity Index (MAI), is strongly prognostic in lymph node‐negative (LNneg) invasive breast cancer. However, its prognostic value has not been compared with the value of Akt and PTEN expression. Material and Methods: Prognostic comparison of Her2Neu, p110alpha (PIK3CA), Akt, mTOR, PTEN, MAI and cell‐cycle regulators in 125 LNneg patients aged <55 years with cyclophosphamide, methotrexate, and 5‐fluorouracil (CMF)‐based adjuvant systemic chemotherapy. Results: Twenty‐one (17%) patients developed distant metastases = DMs (median follow‐up: 134 months). p110alpha correlated (p = 0.01) with pAkt but only in PTEN‐negatives; pAkt correlated (p = 0.02) with mTOR. PTEN‐negativity correlated with high MAI, high grade and ER‐negativity (p = 0.009). The MAI was the strongest prognosticator (Hazard Ratio = HR = 2.9, p = 0.01). Her2Neu/p110α/Akt/mTOR features have no additional prognostic value to the MAI. PTEN had additional value but only in MAI < 3 (39/125 = 31%; 8% DMs). 19/39 = 49% of the MAI < 3 patients have combined MAI < 3 / PTEN+ with 0% DMs, contrasting 15% DMs in MAI < 3 / PTEN− (p = 0.03). Conclusions: In T1−3N0M0 adjuvant CMF‐treated breast cancer patients aged <55 years, MAI was the strongest survival predictor. The PI3K/Akt/mTOR pathway and cell‐cycle regulator characteristics had no additional prognostic value, but PTEN has. Patients with combined MAI < 3 & PTEN‐positivity had 100% survival. The small subgroup of MAI < 3 patients that died were PTEN‐negative.
Population Screening for Colorectal Cancer: Faeces, Endoscopes or X‐Rays?
Authors: Ramsoekh D. , van Leerdam M. E. , van Ballegooijen M. , Habbema J. D. F. , Kuipers E. J.
Published: January 2021

Colorectal carcinoma (CRC) is a common cancer and the second most common cause of death. The therapeutic costs for this disease will continue to rise due to an increasing incidence and the introduction of new chemotherapeutic modalities. Colorectal carcinoma is preceded by precursor lesions, which can be used as a target for early detection and therapy. Biennial population screening with faecal occult blood tests (FOBT) lowers CRC mortality with 14–18%. Five year screening with flexible sigmoidoscopy is a cost‐effective alternative, which yields a higher preventive effect when similar participation rates are achieved. Screening colonoscopy has the advantage of examination of the complete colon but disadvantages are the high participant burden and the higher demand for endoscopic personnel and endoscopy units. Future screening modalities like faecal DNA markers and CT colonography are promising but need further improvement. In Europe, faecal occult blood testing and flexible sigmoidoscopy are currently the most suitable screening modalities for colorectal cancer screening.
Egfr Amplification Specific Gene Expression in Phyllodes Tumours of the Breast
Authors: Agelopoulos Konstantin , Kersting Christian , Korsching Eberhard , Schmidt Hartmut , Kuijper Arno , August Christian , Wülfing Pia , Tio Joke , Boecker Werner , van Diest Paul J. , Brandt Burkhard , Buerger Horst
Published: January 2021

Background: Recently, we were able to show that amplifications of the epidermal growth factor receptor (egfr) gene and the overexpression of EGFR were associated with the initiation and progression of phyllodes tumours. Methods: In order to gain further insights into regulation mechanisms associated with egfr amplifications and EGFR expression in phyllodes tumours, we performed global gene expression analysis (Affymetrix A133.2) on a series of 10 phyllodes tumours, of these three with and seven without amplifications of an important regulatory repeat in intron 1 of egfr (CA‐SSR I). The results were verified and extended by means of immunohistochemistry using the tissue microarray method on an extensively characterized series of 58 phyllodes tumours with antibodies against caveolin‐1, eps15, EGF, TGF‐α, pErk, pAkt and mdm2. Results: We were able to show that the presence of egfr CA‐SSR I amplifications in phyllodes tumours was associated with 230 differentially expressed genes. Caveolin‐1 and eps15, involved in EGFR turnover and signalling, were regulated differentially on the RNA and protein level proportionally to egfr gene dosage. Further immunohistochemical analysis revealed that the expression of caveolin‐1 and eps15 were also significantly correlated with the expression of pAkt (p < 0.05), pERK (p < 0.05), mdm2 (p < 0.01) and EGF (p < 0.001 for caveolin‐1). Eps15 and pERK were further associated with tumour grade (p < 0.01 and p < 0.001, respectively). Conclusion: Our results show that amplifications within regulatory sequences of egfr are associated with the expression of eps15 and caveolin‐1, indicating an increased turnover of EGFR. The interplay between EGFR and caveolin‐1, eps15, pAkt, mdm2 and pERK therefore seems to present a major molecular pathway in carcinogenesis and progression of breast phyllodes tumours.
Intratumor Genetic Heterogeneity of Breast Carcinomas as Determined by Fine Needle Aspiration and TaqMan Low Density Array
Authors: Lyng Maria B. , Lænkholm Anne-Vibeke , Pallisgaard Niels , Vach Werner , Knoop Ann , Bak Martin , Ditzel Henrik J.
Published: January 2021

Background: Gene expression profiling is thought to be an important tool in determining treatment strategies for breast cancer patients. Tissues for such analysis may at a preoperative stage be obtained, by fine needle aspiration (FNA) allowing initiation of neoadjuvant treatment. To evaluate the extent of the genetic heterogeneity within primary breast carcinomas, we examined whether a gene expression profile obtained by FNA was representative of the tumor. Methods: Tumors from 12 consecutive cases of early predominantly estrogen receptor positive (ER+) breast cancer patients undergoing primary surgery were split in halves and FNAs were obtained from each half. A tissue biopsy of the tumors was also snap‐frozen for comparison. Non‐amplified RNA was investigated by the novel qRT‐PCR‐based technique, Low Density Array (LDA) using 4 reference genes and 44 target genes. Results: Comparison of gene expression at the single gene level in the two FNA samples from each tumor demonstrated various degrees of heterogeneity. However, compared as gene expression profiles, intratumor correlations for 9/12 patients were high and these pairs could in a theoretical blinding of all the FNAs be correctly matched by statistical analysis. High correlations between the gene profiles of tumor FNAs and tissue biopsies from the same patient were observed for all patients. A cluster analysis identified clustering of both the two FNAs and the tissue biopsy of the same 9 patients. Conclusion: The overall genetic heterogeneity of breast carcinomas, as sampled by FNA, does not prohibit generation of useful gene profiles for treatment decision making. However, sampling and analysis strategies should take heterogeneity within a tumor, and varying heterogeneity amongst the single genes, into account.
p16 is Consistently Expressed in Endometrial Tubal Metaplasia
Authors: Horree N. , Heintz A. P. M. , Sie-Go D. M. D. S. , van Diest P.J.
Published: January 2021

Background: Cell cycle proteins and HIF‐1α with downstream factors are often abberrantly expressed in (pre)neoplastic tissue. Methods: Paraffin‐embedded specimens of inactive endometrium with TM (n=15), ovarian inclusion cysts (n=6), cervix with TM (tubal metaplasia) (n=3), Fallopian tubes (n=7), cycling endometrium (n=9) and a ciliated cell tumor of the ovary were stained for p16 and LhS28. 39 Endometrioid endometrial carcinomas and 5 serous endometrial carcinomas were stained for p16. Additionally, inactive endometrium (n=15) was immunohistochemically stained for p21, p27, p53, cyclin A, cyclin D1, cyclin E, HIF‐1α, CAIX, Glut‐1 and MIB‐1. Results: A mosaic pattern of expression of p16 was seen throughout in all cases of endometrial TM (15/15), in 2/6 of the ovarian inclusion cysts with TM, in all (3/3) cervical TM and focal in 5/7 of Fallopian tube cases. Mosaic expression was also seen in a ciliated cell tumor of the ovary and in 18/39 of endometrioid endometrial carcinomas, and diffuse p16 expression was seen in 5/5 serous carcinomas. In comparison with normal endometrium, TM areas in the endometrium showed significantly increased expression of HIF‐1α, cyclin E, p21 and cyclin A, and decreased expression of p27. Membranous expression of CAIX and Glut‐1 was only seen in TM areas, pointing to functional HIF‐1α. Conclusion: As p16 is consistently expressed in TM, less and only patchy expressed in the normal Fallopian tube, is paralleled by aberrant expression of cell cycle proteins, HIF‐1α, CAIX and Glut‐1 and resembles the pattern of p16 expression frequently seen in endometrial carcinomas, we propose endometrial TM to be a potential premalignant endometrial lesion.
Increased Expression of the Pro‐Protein Convertase Furin Predicts Decreased Survival in Ovarian Cancer
Authors: Page Robert E. , Klein-Szanto Andrés J. P. , Litwin Samuel , Nicolas Emmanuelle , Al-Jumaily Raid , Alexander Peter , Godwin Andrew K. , Ross Eric A. , Schilder Russell J. , Bassi Daniel E.
Published: January 2021

Background: Proprotein convertases (PCs) are serine proteases that after restricted proteolysis activate many proteins that play a crucial role in cancer such as metalloproteinases, growth factors and growth factor receptors, adhesion molecules, and angiogenic factors. Although the expression of several PCs is increased in many tumors, their expression in primary ovarian tumors has not been studied in detail. We sought to determine if there was an association between the expression of the ubiquitously expressed PCs, furin, PACE‐4, PC‐5 and PC‐7, and ovarian tumor progression. Methods: We assessed their expression by RT‐PCR, Real‐time PCR, Western blot, and immunohistochemistry using cells derived from normal human ovarian surface epithelium (HOSE) and cancer cell lines as well as ovarian epithelial cancer specimens (45 RT‐PCR/Real‐time PCR, and 120 archival specimens for Immunohistochemistry). Results: We found that furin expression was restricted to the cancer cell lines. In contrast, PACE‐4 and PC‐7 showed expression only in normal HOSE cells lines. Furthermore, furin was predominantly expressed in primary tumors from patients who survived for less than five years. The other PCs are either expressed in the group of survivors (PC‐7 and PACE4) or expressed in low amounts (PC‐5). Conclusions: Our studies point to a clear relationship between furin and ovarian cancer. In addition, these results show that furin exhibits the closest association with ovarian cancer among the ubiquitously expressed PCs, arguing against the redundancy of these proteases. In summary, furin may constitute a marker for ovarian tumor progression and could contribute to predict the outcome of this disease.
Stromal Expression of Hypoxia Regulated Proteins Is an Adverse Prognostic Factor in Colorectal Carcinomas
Authors: Cleven Arjen H. G. , van Engeland Manon , Wouters Bradly G. , de Bruïne Adriaan P.
Published: January 2021

Background: Hypoxia modifies the phenotype of tumors in a way that promotes tumor aggressiveness and resistance towards chemotherapy and radiotherapy. However, the expression and influence of hypoxia‐regulated proteins on tumor biology are not well characterized in colorectal tumors. We studied the role of protein expression of hypoxia‐inducible factor (HIF)‐1α, HIF‐2α, carbonic anhydrase 9 (CA9) and glucose transporter 1 (GLUT1) in patients with colorectal adenocarcinomas. Methods: Expression of HIF‐1α, HIF‐2α, CA9 and GLUT1 was quantified by immunohistochemistry in 133 colorectal adenocarcinomas. The expression of hypoxia markers was correlated with clinicopathological variables and overall patient survival. Results: Expression of these hypoxia markers was detected in the epithelial compartment of the tumor cells as well as in tumor‐associated stromal cells. Although tumor cells frequently showed expression of one or more of the investigated hypoxia markers, no correlation among these markers or with clinical response was found. However, within the tumor stroma, positive correlations between the hypoxia markers HIF‐2α, CA9 and GLUT1 were observed. Furthermore expression of HIF‐2α and CA9 in tumor‐associated stroma were both associated with a significantly reduced overall survival. In the Cox proportional hazard model, stromal HIF‐2α expression was an independent prognostic factor for survival. Conclusion: These observations show, that expression of hypoxia regulated proteins in tumor‐associated stromal cells, as opposed to their expression in epithelial tumor cells, is associated with poor outcome in colorectal cancer. This study suggests that tumor hypoxia may influence tumor‐associated stromal cells in a way that ultimately contributes to patient prognosis.
Chromosomal Genotype in Breast Cancer Progression: Comparison of Primary and Secondary Manifestations
Authors: Friedrich Katrin , Weber Theresa , Scheithauer Jens , Meyer Wolfdietrich , Haroske Gunter , Kunze Klaus Dietmar , Baretton Gustavo
Published: January 2021

The purpose of this study was to compare the chromosomal genotype between breast cancers with and without secondary manifestations and between primary tumors and their secondary manifestations. Eighty six breast cancers, twenty lymph node metastases, ten distant metastases and ten local recurrences were analyzed by comparative genomic hybridization. Tumors with local recurrences showed significant more frequent losses at 2q32 than the tumors without recurrences. Lymph node positive cases showed significant more frequent losses at 9p21 than node negative cases. Lymph node metastases exhibited significant more frequent losses at 7q11, 14q24.3–q31 and 17q22–q24 than their primary tumors. In cases with distant metastases, losses at 5q23 were more frequent than in those without, but not reaching the significance level. The distant metastases showed significant more frequent losses at 5p15, 12q24 and 17q22–q24 than the primary tumors. These results reveal strong evidence that the potential for progression is determined in the primary tumor and that different ways of the development of local recurrences, lymph node and distant metastases exist. After confirmation of the results by interphase FISH on tissue micro arrays, the detection of these specific chromosomal imbalances may contribute to a more individual prediction of prognosis in breast cancer.
Immunohistochemical Detection and Localization of Somatostatin Receptor Subtypes in Prostate Tissue from Patients with Bladder Outlet Obstruction
Authors: Montironi Rodolfo , Cheng Liang , Mazzucchelli Roberta , Morichetti Doriana , Stramazzotti Daniela , Santinelli Alfredo , Moroncini Gianluca , Galosi Andrea B. , Muzzonigro Giovanni , Comeri Giancarlo , Lovisolo Jon , Cosciani-Cunico Sergio , Bono Aldo V.
Published: January 2021

Background and Aim of the Study: Scant information on the cellular distribution of the five somatostatin receptor (SSTR) subtypes in the normal prostate and in neoplasms of the prostate has been reported in very few studies in which techniques, such as in situ hybridization histochemistry, autoradiography, and more recently immunohistochemistry, have been applied. The aim of the study was to examine immunohistochemically the distribution and localization of these 5 subtypes in the various tissue components in normal prostate.

Materials: The study was conducted in 14 surgical specimens of normal prostate tissue from adenomectomy specimens from patients with bladder outlet obstruction. The distribution and localization of the 5 somatostatin receptor (SSTR) subtypes was investigated with an immunohistochemical technique. Specificity of the antibodies against the 5 receptor subtypes was preliminarily investigated.

Results: Close to 90% of secretory cells showed a weak positivity in the cytoplasm, the proportion ranging from 86.3% (SSTR4) to 89.9% (SSTR5). Strong immunoreactivity was seen in a small proportion of cells, ranging from 0.8% (SSTR3) to 3.2% (SSTR1). For the subtypes 1 and 3 the greatest proportion of basal cells showed a moderate intensity (42.5 and 41.4%, respectively), strong immunoreactivity being observed only in 18.1 and 15.8% of cells, respectively. For the subtypes 2, 4 and 5, the majority of cells showed a weak intensity (72.3, 65.7 and 65.1%, respectively). Subtype 1 showed a strong immunoreactivity in the cytoplasm in 60% of the smooth muscle cells. With subtypes 2, 3 and 4 the greatest proportion of cells showed a weak intensity (63.4, 89.8 and 81.7%, respectively). With the subtype 5 the majority of cells (59.8%) were negative. Subtype 1 showed a strong immunoreactivity in the cytoplasm in 98.6% of the endothelial cells. With subtypes 3 and 4 the greatest proportion of cells showed a weak intensity (73.5 and 56.4%, respectively). With the subtype 2 and 5 the majority of cells were negative (59.1 and 50.7%, respectively).

Conclusions: Our immunohistochemical study on the SSTRs expands our knowledge in the distribution of these subtypes in the
various tissue components in the prostate. Such an information may prove useful in developing further non-surgical strategies for the prevention and treatment of benign prostatic hyperplasia and, in particular, of preneoplastic and neoplastic lesions of the prostate.
PRIMA-1MET Inhibits Growth of Mouse Tumors Carrying Mutant p53
Authors: Zache Nicole , Lambert Jeremy M. R. , Wiman Klas G. , Bykov Vladimir J. N.
Published: January 2021

Reactivation of the tumor suppressor activity to mutant p53 should trigger massive apoptosis and eliminate tumors. The low molecular weight compounds PRIMA-1 and the structural analog PRIMA-1MET reactivate human mutant p53 in vitro and suppress growth of human tumor xenografts in SCID mice. However, little is known about their effect on mouse mutant p53 in mouse tumor cells. We have examined the effect of PRIMA-1MET on mouse sarcomas, mammary carcinomas and chemically induced fibrosarcomas. PRIMA-1MET showed potent growth suppression in mutant p53-carrying mouse tumors in vitro and a significant anti-tumor effect in syngeneic mice in vivo. These results demonstrate that PRIMA-1MET targets mouse tumors carrying mutant p53 and provide strong support for the anti-tumor efficiency of PRIMA-1METin vivo.
MLPAnalyzer: Data Analysis Tool for Reliable Automated Normalization of MLPA Fragment Data
Authors: Coffa Jordy , van de Wiel Mark A. , Diosdado Begoña , Carvalho Beatriz , Schouten Jan , Meijer Gerrit A.
Published: January 2021

Background: Multiplex Ligation dependent Probe Amplification (MLPA) is a rapid, simple, reliable and customized method for detection of copy number changes of individual genes at a high resolution and allows for high throughput analysis. This technique is typically applied for studying specific genes in large sample series. The large amount of data, dissimilarities in PCR efficiency among the different probe amplification products, and sample-to-sample variation pose a challenge to data analysis and interpretation. We therefore set out to develop an MLPA data analysis strategy and tool that is simple to use, while still taking into account the above-mentioned sources of variation.

Materials and Methods: MLPAnalyzer was developed in Visual Basic for Applications, and can accept a large number of file formats directly from capillary sequence systems. Sizes of all MLPA probe signals are determined and filtered, quality control steps are performed, and variation in peak intensity related to size is corrected for. DNA copy number ratios of test samples are computed, displayed in a table view and a set of comprehensive figures is generated. To validate this approach, MLPA reactions were performed using a dedicated MLPA mix on 6 different colorectal cancer cell lines. The generated data were normalized using our program and results were compared to previously performed array-CGH results using both statistical methods and visual examination.

Results and Discussion: Visual examination of bar graphs and direct ratios for both techniques showed very similar results, while the average Pearson moment correlation over all MLPA probes was found to be 0.42. Our results thus show that automated MLPA data processing following our suggested strategy may be of significant use, especially when handling large MLPA data sets, when samples are of different quality, or interpretation of MLPA electropherograms is too complex. It remains, however, important to recognize that automated MLPA data processing may only be successful when a dedicated experimental setup is also considered.
HA14-1 is Able to Reconstitute the Impaired Mitochondrial Pathway of Apoptosis in Renal Cell Carcinoma Cell Lines
Authors: Heikaus Sebastian , van den Berg Linda , Kempf Tobias , Mahotka Csaba , Gabbert Helmut Erich , Ramp Uwe
Published: January 2021

Renal cell carcinomas (RCCs) exhibit a marked resistance towards apoptosis. Although most apoptotic stimuli converge at the level of the mitochondria, little is known about the mitochondrial apoptosis pathway in renal cell carcinomas. The aim of the present study, therefore, was to investigate the functionality of the mitochondrial apoptosis pathway in renal cell carcinoma cell lines by exposure to TRAIL, etoposide, HA14-1 and betulinic acid activating the mitochondria by different mechanisms. Sensitivity to TRAIL-induced apoptosis correlated with cleavage of the initiator caspase-8, but the mitochondrial apoptosis pathway was not induced. Similarly, etoposide and betulinic acid could not induce mitochondrial damage. In contrast, HA14-1 was able to activate mitochondrial apoptosis, thereby demonstrating functionally inducible signalling pathways downstream of the mitochondria. The intactness of the pathways upstream of the mitochondria was shown by pretreatment of TRAIL-sensitive cell lines with HA14-1, which could reconstitute TRAIL-induced mitochondrial damage and resulted in a synergistic apoptosis induction.

Our results demonstrate that the apoptotic pathways upstream and downstream of the mitochondria are intact and inducible in renal cell carcinoma cell lines. However, resistance towards mitochondrial apoptosis is located on the level of the mitochondria themselves.
A Fast, Sensitive and Accurate High Resolution Melting (HRM) Technology-Based Assay to Screen for Common K-ras Mutations
Authors: Kramer D. , Thunnissen F. B. , Gallegos-Ruiz M. I. , Smit E. F. , Postmus P. E. , Meijer C. J. L. M. , Snijders P.J.F. , Heideman D. A. M.
Published: January 2021

Background: Increasing evidence points to a negative correlation between K-ras mutations and patient’s response to, or survival benefit after, treatment with EGFR-inhibitors. Therefore, rapid and reliable assays for mutational analysis of the K-ras gene are strongly needed.

Methods: We designed a high resolution melting (HRM) technology-based approach followed by direct sequencing to determine K-ras exon 1 (codons 12/13) tumour genotype.

Results: Reconstruction experiments demonstrated an analytical sensitivity of the K-ras exon 1 HRM assay following sequencing of 1.5–2.5% of mutated DNA in a background of wild-type DNA. Assay reproducibility and accuracy were 100%. Application of the HRM assay following sequencing onto genomic DNA isolated from formalin-fixed paraffin-embedded tumour specimens of non-small cell lung cancer (n=91) and colorectal cancer (n=7) patients revealed nucleotide substitutions at codons 12 or 13, including a homozygous mutation, in 33 (34%) and 5 (5%) cases, respectively. Comparison to conventional nested-PCR following cycle-sequencing showed an overall high agreement in genotype findings (kappa value of 0.96), with more mutations detected by the HRM assay following sequencing.

Conclusion: HRM allows rapid, reliable and sensitive pre-screening of routine diagnostic specimens for subsequent genotyping of K-ras mutations, even if present at low abundance or homozygosity, and may considerably facilitate personalized therapy planning.
LOH at 1p31 (ARHI) and Proliferation in Lymph Node-Negative Breast Cancer
Authors: Janssen Emiel A. M. , Øvestad Irene T. , Skaland Ivar , Søiland Håvard , Gudlaugsson Einar , Kjellevold Kjell H. , Nysted Arne , Søreide Jon-Arne , Baak Jan P. A.
Published: January 2021

Background: The mitotic activity index (MAI) is a strong prognosticator in node-negative invasive breast cancer patients. Recently, a correlation between the MAI and specific chromosomal aberrations at chromosome 1p was described.

Methods: Analysis of MAI, immunohistochemical staining patterns for proliferation-associated phosphohistone H3 (PPH3), phosphorylated ERK1/2, p21, cyclin E, Ki67 and cyclin D1 proteins; and prognosis in 158 adjuvant chemotherapy-treated T1-2N0M0 invasive breast cancer patients, analysis of LOH at 1p31 (including ARHI) using the dinucleotide repeats D1S207, D1S430 and D1S464 in 76 patients. Single and multivariate survival analysis was used to evaluate the importance of the various markers tested.

Results: LOH at 1p31 did not correlate with MAI nor provide prognostic information. Phosphohistone H3 was the best prognosticator for patients in all age groups with 20 year distant metastasis free survival of distant metastases 93% vs. 72% respectively (p = 0.004, HR = 4.5). In multivariate analysis, phosphohistone H3 < 13 vs. ≥13 exceeded the prognostic value of the mitotic activity index.

Conclusions: LOH at 1p31 is common in breast cancer, and correlates with loss of proliferation-associated proteins, but not with MAI, PPH3 or prognosis. PPH3 is the best prognosticator in this study group of adjuvant chemotherapy-treated lymph node-negative breast cancer patients.
Basic Fibroblast Growth Factor-Mediated Overexpression of Vascular Endothelial Growth Factor in 1F6 Human Melanoma Cells is Regulated by Activation of PI-3K and p38 MAPK
Authors: Fontijn Dennis , Bosch Linda J. W. , Duyndam Monique C. A. , van Berkel Maria P. A. , Janmaat Maarten L. , Boven Epie
Published: January 2021

Background: 1F6 human melanoma xenografts overexpressing either the 18 kD (18kD) form or all (ALL) forms of human basic fibroblast growth factor (bFGF) demonstrate an abundant number of microvessels and accelerated growth. We now examined whether bFGF mediates vascular endothelial growth factor (VEGF) expression.

Methods: Quantitative RT-PCR was used to determine bFGF and VEGF mRNA, VEGF protein secretion was measured by ELISA and VEGF promoter activation was assessed by a dual luciferase activity assay. Western blot was carried out to detect phosphorylation of bFGF-regulated target proteins.

Results: In 1F6-18kD and 1F6-ALL clones VEGF mRNA was increased 4- to 5-fold and VEGF protein secretion was highly stimulated due to activation of the VEGF promotor. PI-3K, p38 MAPK and ERK1/2 MAPK pathways were activated, while inhibition of PI-3K or p38 resulted in, respectively, 55% and up to 70% reduction of VEGF mRNA overexpression. A concurrent 60% decrease in VEGF protein secretion was mostly apparent upon inhibition of PI-3K. Inhibition of ERK1/2 hardly affected VEGF mRNA or protein secretion. Two unselected human melanoma cell lines with high metastatic potential contained high bFGF and VEGF, while three non- or sporadically metastatic cell lines displayed low bFGF and VEGF.

Conclusion: These data indicate that stimulation of VEGF protein secretion in response to bFGF overexpression may contribute to increased vascularization and enhanced aggressiveness in melanoma.
Prognostic Relevance of Promoter Hypermethylation of Multiple Genes in Breast Cancer Patients
Authors: Sharma Gayatri , Mirza Sameer , Yang Yi-Hsin , Parshad Rajinder , Hazrah Priya , Gupta Siddartha Datta , Ralhan Ranju
Published: January 2021

Background: Methylation-mediated suppression of detoxification, DNA repair and tumor suppressor genes has been implicated in cancer development. This study was designed to investigate the impact of concurrent methylation of multiple genes in breast tumors on disease prognosis.

Methods: Methylation specific PCR was carried out to analyze the methylation status of seven genes in archived breast tissues and determine the effect of aberrant methylation of multiple genes on disease prognosis and patients’ survival.

Results: Promoter hypermethylation was observed in PRB 67%, ERα 64%, RASSF1A 63%, p16INK4A 51%, PRBβ2 22%, GSTP1 25% and BRCA1 27% of the breast cancers, respectively. Concurrent methylation of BRCA1, ERα, GSTP1 and RARβ2, was observed in a large proportion of breast cancers analyzed, suggesting that these genes do not appear to be methylated alone. Patients with high methylation indices had poor prognosis (p < 0.001, Hazards ratio = 14.58). Cox regression analysis showed RARβ2 promoter methylation to be an independent important determinant of breast cancer prognosis.

Conclusions: Our results suggest that methylation of multiple genes plays an important role in prognosis of breast cancer. Our study not only describes the association of methylation mediated silencing of multiple genes with the severity of disease, but also drives to speculate the molecular crosstalk between genes or genetic pathways regulated by them individually.
Cutaneous Human Papillomavirus E7 Type-Specific Effects on Differentiation and Proliferation of Organotypic Skin Cultures
Authors: Westphal K. , Akgül B. , Storey A. , Nindl I.
Published: January 2021

Background: A role for cutaneous human β-papillomavirus (HPV) types as co-factors in the development of non-melanoma skin cancer has been postulated. Here we have investigated the effects of E7 expression on keratinocyte differentiation, proliferation and cell-cycle proteins in organotypic skin cultures.

Methods: Recombinant retroviruses containing the E7 genes from cutaneous HPV types 1, 4, 5, 8, 20, 38 and RTRX7 were produced that include types associated with benign and malignant lesions. Adult human primary keratinocytes were transduced with these recombinant retroviruses and differentiated into skin-equivalents using de-epidermalised human dermis.

Results: Expression patterns of the basal keratinocyte marker cytokeratin 14 (CK14) were not altered by any of the viral E7 types analysed. However, expression of the early and late differentiation markers CK10 and involucrin were markedly altered in HPV 1, 4 and 38 cultures. The highest proliferation rates in basal cell layers, as judged by BrdU and Ki67 staining, were observed in HPV 1, 4 and 38 cultures. Interestingly, co-expression of cyclin E and p16INK4a within the same cell of the suprabasal cell layers was observed only in cultures generated using E7 of HPV 5 or HPV 8.

Conclusion: HPV types associated with either benign or malignant lesions perturb keratinocyte proliferation and differentiation in different ways. Moreover, expression of E7 from HPV 5 or HPV 8 seem able to overcome p16INK4a induced cell cycle arrest in a subset of keratinocytes.
The Prognostic Value of the Proliferation Marker Phosphohistone H3 (PPH3) in Luminal, Basal-Like and Triple Negative Phenotype Invasive Lymph Node-Negative Breast Cancer
Authors: Skaland Ivar , Janssen Emiel A. M. , Gudlaugsson Einar , Guo Lydia Hui Ru , Baak Jan P. A.
Published: January 2021

Purpose: Prognostic comparison of phosphohistone-H3 (PPH3) with Cytokeratin 5/6 and/or 14 positive (= basal-CK), triple (ER, PR, HER2)-negative (= TNP) and basal-like (= TNP and basal-CK positive) phenotype in invasive breast cancers.

Patients and Methods: Classical variables, PPH3, ER, PR, basal-CK and HER2 in 240 T1–2N0M0 patients under 71 years.

Results: TNP and basal-like cancers had higher PPH3 expression than the other cancers (mean 48 versus 11, P < 0.001). Fifteen percent of the patients in the whole group, but 32–38% of TNP and basal-like cancers recurred.With multivariate analysis, PPH3 < 13 (n = 156) versus ≥ 13 (n = 84 = 35% of all cases) was the strongest and only prognosticator (10-year survival 96% and 64%, P ≤ 0.001, Hazard ratio = 9.0).

Conclusion: PPH3 is the strongest prognosticators in luminal, Triple negative and basal-like T1–2N0M0 invasive breast cancers.
Expression of ERα, ERβ and Co-Regulator PELP1/MNAR in Colorectal Cancer: Prognostic Significance and Clinicopathologic Correlations
Authors: Grivas Petros D. , Tzelepi Vassiliki , Sotiropoulou-Bonikou Georgia , Kefalopoulou Zinovia , Papavassiliou Athanasios G. , Kalofonos Haralabos
Published: January 2021

Background: Estrogen receptor β (ERβ) is abundantly expressed in colorectal tissue, but its role in colorectal carcinogenesis remains elusive. ER novel co-regulator, proline-, glutamic acid- and leucine-rich protein 1 (PELP1/MNAR) has been characterized, but its expression in colorectal carcinomas has not been investigated.

Methods: ERα, ERβ and PELP1/MNAR protein expression were evaluated by immunohistochemistry in colorectal normal mucosa, adenomas and adenocarcinomas from 113 patients with colorectal cancer.

Results: ERα expression is extremely rare in colorectal tissue and its expression does not appear to be associated with colorectal carcinogenesis. ERβ and PELP1/MNAR were detected in the nucleus of epithelial, endothelial, inflammatory, smooth muscle cells and myofibroblasts. When intensity of staining was taken into account, the expression of both proteins was significantly increased in epithelial cells of carcinomas compared to normal mucosa. ERβ expression in epithelial cells was correlated with decreased disease progression – free survival. PELP1/MNAR overexpression in epithelial cells was found to be an independent favorable prognostic factor. Additionally, the expression of both proteins was significantly increased in stromal myofibroblasts of carcinomas compared to adenomas and normal mucosa.

Conclusion: ERβ and PELP1/MNAR appear to be involved in colorectal tumorigenesis and might have prognostic significance.
Functional Implication of Netrin Expression in Malignant Melanoma
Authors: Kaufmann Simone , Kuphal Silke , Schubert Thomas , Bosserhoff Anja K.
Published: January 2021

Background: Malignant melanoma cells are known to have altered expression of genes supporting proliferation and invasion, however, the expression of molecules of the Netrin family of repellent factors has not been analyzed in melanomas until now.

Results: Here, we show that Netrin-1 expression is strongly induced in melanoma cells compared to melanocytes in vivo and in vitro controlled at the transcriptional level via ETS-1. In addition, the expression of the netrin receptor UNC5B was induced and that of UNC5C was reduced in the tumor cells. In order to determine the functional relevance of Netrin-1 expression in malignant melanoma, Netrin expression in melanoma cells was reduced by siRNA attempts and primary human melanocytes were treated with recombinant Netrin-1. The cells showed no changes in proliferation or apoptosis, however, a strong reduction of migratory properties was observed in the melanoma cells after reduction of Netrin expression whereas melanocyte migration was strongly induced by treatment with Netrin.

Conclusions: Our study suggests that Netrin-1 promotes melanoma cell invasion and migration and therefore has an important role in the progression of malignant melanoma.