Marker For Malignant Cells Research Articles

Diagnostic and Prognostic Importance of AgNOR Pleomorphism in Cervical Carcinoma

Nucleolar organizer region-associated proteins are argyrophilic and visualized by silver staining called AgNOR. It can be used as a diagnostic and prognostic marker for malignant cells which exhibit a greater AgNOR protein. The present study aimed to evaluate the correlation of AgNOR counts with severity and prognosis of cervical lesion. Silver nitrate staining was performed in a total of 50 cervical smears collected from the women coming to the outpatient department of the hospital diagnosed as cytological normal, inflammatory, low grade squamous intraepithelial lesion, high grade squamous intraepitelial lesion and squamous cell carcinoma (SCC). Total cell counts were determined for single and pleomorphic dots. Both single and pleomorphic dots increase with an increase in grades of lesions from cytological normal to SCC. But mean cell counts of pleomorphic dots increase with cytological abnormality. Correlation of disease severity with a cell count of pleomorphic dots (r = 0.96, p ≤ 0.001) and single dots (r = 0.98, p ≤ 0.001) is positive and significant. A significant increase in pleomorphic dot in patients indicated the prognostic importance of pleomorphic counts during the increase in cytological abnormalities of cervical lesions.

ScTyper: a comprehensive pipeline for the cell typing analysis of single-cell RNA-seq data

BackgroundRecent advances in single-cell RNA sequencing (scRNA-seq) technology have enabled the identification of individual cell types, such as epithelial cells, immune cells, and fibroblasts, in tissue samples containing complex cell populations. Cell typing is one of the key challenges in scRNA-seq data analysis that is usually achieved by estimating the expression of cell marker genes. However, there is no standard practice for cell typing, often resulting in variable and inaccurate outcomes.ResultsWe have developed a comprehensive and user-friendly R-based scRNA-seq analysis and cell typing package, scTyper. scTyper also provides a database of cell type markers, scTyper.db, which contains 213 cell marker sets collected from literature. These marker sets include but are not limited to markers for malignant cells, cancer-associated fibroblasts, and tumor-infiltrating T cells. Additionally, scTyper provides three customized methods for estimating cell-type marker expression, including nearest template prediction (NTP), gene set enrichment analysis (GSEA), and average expression values. DNA copy number inference method (inferCNV) has been implemented with an improved modification that can be used for malignant cell typing. The package also supports the data preprocessing pipelines by Cell Ranger from 10X Genomics and the Seurat package. A summary reporting system is also implemented, which may facilitate users to perform reproducible analyses.ConclusionsscTyper provides a comprehensive and user-friendly analysis pipeline for cell typing of scRNA-seq data with a curated cell marker database, scTyper.db.

Minichromosome Maintenance Protein Expression According to the Grade of Atypism in Actinic Keratosis

The minichromosome maintenance (MCM) family is a group of proteins that are key initiation factors for DNA replication and are expressed only in cycling cells. Recent studies on various cancerous conditions have shown that MCM proteins are better markers for malignant cells compared to other proliferative markers. It has been also proven that MCM proteins are independent prognostic factors. The aim of this study was to characterize the pattern and frequency of MCM 2 protein expression in actinic keratosis (AK) and determine whether the expression is correlated with the degree of histological atypism. Biopsy samples of 34 patients who had been diagnosed as AK were used in this study. Samples were divided into three groups (grade I, grade II, and grade III) according to the degree of atypism. Immunohistochemical staining for MCM 2 protein, Ki-67, and proliferating cell nuclear antigen was performed, and the number of positively staining cells per unit area (10⁻⁴ μm²) was calculated for evaluation of immunoreactivity. MCM 2 protein was expressed in atypical keratinocytes in AK. Mean numbers of immunoreactive cells positive for MCM 2 were 165.1 in grade I, 304.5 in grade II, and 513.3 in grade III. Moreover, the correlation between the immunoreactivity for MCM 2 protein and AK grade was significantly more positive than that for other markers. Thus, we suggest that MCM 2 protein is a reliable marker for diagnosing and grading AK and further could be hypothesized as an important prognostic factor.

Frequent Instability of Type I and II Aberrations Between Diagnosis and Relapse Observed in Pediatric Aml; Implications for Targeted Therapy and MRD Detection.

Abstract 1624Poster Board I-650Activating type I mutations provide cells with proliferative and survival advantages. Together with type II abnormalities, which cause a differentiation arrest and an increase in self-renewal properties, they cooperate to cause acute myeloid leukemia (AML). Currently, many novel drugs have been developed to specifically target these mutations and the mutations are also used as marker of malignant cells in MRD detection. It is thus of importance to accurately determine the mutation status of AML samples. Our previous study showed frequent shifts in FLT3/ITD mutations between initial and relapse samples. Losing an FLT3/ITD mutation at relapse was associated with prolonged time to relapse, whereas acquiring an FLT3/ITD at relapse was associated with a shorter time to relapse (Cloos et al. Leukemia, 2006). We now extended the mutation analysis of paired initial and relapsed pediatric samples to study the stability of type I and II mutations including RAS oncogenes (e.g. N-RAS, K-RAS), receptor tyrosine kinases (FLT3 and KIT), WT1, NPM1, PTPN11 and CEBPá genes.Samples were analyzed of 70 pediatric AML patients who were treated with protocols of the BFM-AML Study Group or Dutch Childhood Oncology Group between 1992 and 2004. Using capillary gel electrophoresis based fragment analysis, we analyzed the patient samples for insertions/deletions in exons 14, 15 and 20 of FLT3, exon 11 of KIT, exon 12 of NPM1 and 3 hotspots in the CEBPá gene. FRET based melting curve analysis or high resolution melting curve analysis were used to detect point mutations in exons 8, 9 and 17 of KIT, exons 3 and 13 of PTPN11, codon 12/13 and 61 mutations of N-RAS and codon 12/13 mutations of K-RAS. Exons 7,8,9 and 10 of WT1 were screened using multiplex ligation probe amplification.Instabilities were found in 25 out of 70 (36%) patients (Table 1). In none of the cases a mutation at diagnosis predicted time to relapse. However, when patients were stratified according to the presence of a type I/II mutation at relapse, independent of its presence at diagnosis, patients who had a FLT3/ITD or RAS mutation at relapse, experienced a significantly shorter mean time to relapse than patients without a mutation at relapse (Table 2).One possible explanation for the observed shifts in type I/II aberrations is the expansion at relapse of minor leukemic sub-clones, present, but not detected at diagnosis. Of a patient of whom we had enough cryopreserved cells from diagnosis samples available to FACSsort small amounts of cells (<25) from different primitive leukemic blast compartments. We found that the malignant progenitor like CD133-CD34+CD38dim fraction contained a clone with the type I/II molecular profile which was present at relapse but was not detected in the bulk of the diagnosis sample (Figure 1).In conclusion, in a relatively large cohort of paired diagnosis-relapse samples, we established that a high percentage (nearly 40%) of pediatric AML patients show changes in type I/II mutations, with both gains and losses from diagnosis to relapse. This indicates that mutation profiling at diagnosis is often not sufficient for accurate relapse risk assessment. Additional molecular characterization of AML at minimal residual disease will guide targeted therapy. The mutational shifts can be explained by the expansion of minor leukemic sub-clones during evolution of the disease. Although we show clonal selection in one case we cannot rule out the possibility that in other patients the observed shifts are due to the development of new leukemic sub-clones which are induced during therapy and related to genomic instability. To elucidate this, future research will focus on the detection and characterization of malignant sub-clones at different stages of the disease.Financially supported by Dutch Cancer Society (VU 2005-3666)Table 1Frequency of mutation shifts in genes of interest for 70 paired pediatric AML samplesGene of interestFLT3/ITDFLT3 D835RASWT1KITPTPN11NPM1CEBPáGain21450001Loss41700110Table 2Association between mean time to relapse and mutation statustime of analysismean time to relapse (months, neg vs. pos)ANOVA p valueFLT3/ITDdiagnosis16.0 vs 17.20.8relapse17.8 vs 7.80.05RASdiagnosis18.0 vs 11.20.1relapse18.4 vs 9.40.01WT1diagnosis17.5 vs 15.10.7relapse18.6 vs 12.50.28 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CELLULAR DIFFERENTIATION DETERMINES THE EXPRESSION OF THE HYPOXIA-INDUCIBLE PROTEIN NDRG1 IN PANCREATIC CANCER

N-myc downstream-regulated gene-1 (NDRG1) is a recently described hypoxia-inducible protein that is upregulated in various human cancers. Pancreatic ductal adenocarcinoma, called pancreatic cancer, is a highly aggressive cancer that is characterised by its avascular structure, which results in a severe hypoxic environment. In this study, we investigated whether NDRG1 is upregulated in these tumours, thus providing a novel marker for malignant cells in the pancreas. By immunohistochemistry, we observed that NDRG1 was highly expressed in well-differentiated cells of pancreatic cancer, whereas the poorly differentiated tumour cells were negative. In addition, hyperplastic islets and ducts of nonquiescent pancreatic tissue were positive. To further explore its selective expression in tumours, two well-established pancreatic cancer cell lines of unequal differentiation status were exposed to 2% oxygen. NDRG1 mRNA and protein were upregulated by hypoxia in the moderately differentiated Capan-1 cells; however, its levels remained unchanged in the poorly differentiated Panc-1 cell line. Taken together, our data suggest that NDRG1 will not serve as a reliable marker of tumour cells in the pancreas, but may serve as a marker of differentiation. Furthermore, we present the novel finding that cellular differentiation may be an important factor that determines the hypoxia-induced regulation of NDRG1.

Cellular differentiation determines the expression of the hypoxia-inducible protein NDRG1 in pancreatic cancer

N-myc downstream-regulated gene-1 (NDRG1) is a recently described hypoxia-inducible protein that is upregulated in various human cancers. Pancreatic ductal adenocarcinoma, called pancreatic cancer, is a highly aggressive cancer that is characterised by its avascular structure, which results in a severe hypoxic environment. In this study, we investigated whether NDRG1 is upregulated in these tumours, thus providing a novel marker for malignant cells in the pancreas. By immunohistochemistry, we observed that NDRG1 was highly expressed in well-differentiated cells of pancreatic cancer, whereas the poorly differentiated tumour cells were negative. In addition, hyperplastic islets and ducts of nonquiescent pancreatic tissue were positive. To further explore its selective expression in tumours, two well-established pancreatic cancer cell lines of unequal differentiation status were exposed to 2% oxygen. NDRG1 mRNA and protein were upregulated by hypoxia in the moderately differentiated Capan-1 cells; however, its levels remained unchanged in the poorly differentiated Panc-1 cell line. Taken together, our data suggest that NDRG1 will not serve as a reliable marker of tumour cells in the pancreas, but may serve as a marker of differentiation. Furthermore, we present the novel finding that cellular differentiation may be an important factor that determines the hypoxia-induced regulation of NDRG1.

Diagnostic DNA-flow- vs. -image-cytometry in effusion cytology.

Aims: To determine the sensitivity and specificity of flow‐ and image‐cytometry for the detection of DNA‐aneuploidy as a marker for malignant cells in effusions. Methods: 200 effusions (80 tumor cell‐positive, 74 negative and 46 cytologically equivocal) were stained with DAPI‐SR for DNA‐flow‐ and with Feulgen‐Pararosaniline for ‐image‐cytometry. They were measured using a PAS‐flow‐cytometer and an AutoCyte‐QUIC‐DNA‐workstation according to the ESACP consensus reports for DNA‐flow‐ and ‐image‐cytometry, respectively [7,23,29,49]. Results: Sensitivity of DNA‐aneuploidy for the identification of malignant cells was 32.1% for DNA‐flow‐ and 75.0% for ‐image‐cytometry, specificity of ‐euploidy in benign cells was 100.0% for both methods. Positive predictive value of DNA‐aneuploidy for the identification of malignant cells was 100.0% for both techniques, negative predictive value of DNA‐euploidy was 48.6% for DNA‐flow‐ and 72.0% for ‐image‐cytometry. Conclusions: Searching for DNA‐aneuploidy as a diagnostic marker for neoplastic cells in serous effusions image‐cytometry revealed superior sensitivity as compared with monoparametric flow cytometry.

HLA‐G protein expression is not induced during malignant transformation

To evaluate the biological relevance of HLA-G expression during tumoral transformation, we analyzed its expression in different malignant cells and immune effector cells infiltrating solid tumors. Our analysis of 33 tumor cell lines and 53 tumoral biopsies demonstrated that: i) six tumor cell lines display HLA-G transcription with differential alternative splicing patterns and only Jeg3 choriocarcinoma and MCF-7 breast adenocarcinoma cell lines express HLA-G translated products; and ii) HLA-G antigens are not expressed in malignantly transformed cells derived from lung (n=18), liver (n=5), colon (n=5), breast (n=10), kidney (n=5), ovary (n=5), and larynx (n=5) tissues ex vivo. The healthy tissues surrounding these tumor tissues do not express HLA-G molecules either. On the other hand, surprisingly, HLA-G products were detected in activated macrophages and dendritic cells localized in tumoral biopsies of 5 out of 18 different lung carcinomas. No HLA-G labelling was observed in resident mononuclear phagocytes of surrounding healthy tissues. Our observations clearly demonstrate that HLA-G is not a marker of malignant cells but appears as a gene expressed in tumor-associated macrophages and dendritic cells, preferentially in those recruited in lung carcinomas. Our findings suggest that specific environmental factors around lung tumors could be involved in the induction of HLA-G protein expression.

Interphase FISH: a rapid method for detecting malignant plasma cells in multiple myeloma patients submitted to autologous transplantation.

Fluorescence in situ hybridization (FISH) on interphase nuclei has been shown to be an efficient method for detecting aneuploidy in multiple myeloma (MM). The aim of this study was to test the feasibility of FISH techniques for detecting malignant cells in the harvests of MM patients submitted to autologous transplantation. As trisomy 9 (T9) is a frequent event in MM, we used it as a genetic marker of malignant plasma cells. T9 was detected in 45 out of 55 MM bone marrow samples (81.8%) using a chromosome 9 centromeric (C9C) probe. Twenty-four of the 55 MM patients were subjected to high-dose therapy followed by autologous unselected progenitor cell transplantation. Trisomy 9 was detected in 20 patients and was used as a marker of malignant cells. Upon karyotypic analysis, three of the four remaining patients without T9 showed an unbalanced translocation leading to a complete trisomy of the long arm of chromosome 1 (T1q). We thus used a 1q juxtacentromeric probe, pUC1.77, as another genetic marker of malignant plasma cells in these three further patients. FISH with C9C or pUC1.77 probes was performed on the harvests of these 23 patients and detected clonal cells in 11 transplants. The disease-free survival from graft was significantly longer for the patients who had no malignant cells in their transplant (P=0.009). The median disease-free survival was 23 months in these patients, as compared to 12 months in the patients whose transplant was contaminated. As almost all MM are cytogenetically abnormal, FISH with adequate probes represents a simple, quantitative tool for rapid detection of malignant cells in the harvests. Our results also suggest that the presence of MM cells in the transplant may be predictive of poor outcome.

Thymidine kinase expression: A marker for malignant cells

The expression of thymidine kinase--an enzyme of the DNA precursor pathway--is strictly regulated during the normal cellular cycle, but is much higher and permanently expressed in malignant growing cells. Using a new cytofluorometric assay for thymidine kinase in single cells, we were able to discriminate between normal growing cells and virally transformed cells or lines derived from tumours. In material (blood and bone marrow) taken from leukaemia patients, we identified the leukaemic cells in a surplus of normal leucocytes. From cell cultures representing a tumour progression model, only the later, and malignant, stages showed enhanced fluorescence, whereas benign tumour cells looked normal.

Differential expression of jackfruit-lectin-specific glycoconjugates in metastatic adenocarcinoma and reactive mesothelial cells-a diagnostic aid in effusion cytology.

Distinguishing reactive mesothelial cells from adenocarcinoma cells in serous effusions on the basis of morphological criteria alone is often difficult. Interest has therefore been focused on identifying reliable methods to supplement the conventional cytological techniques. Plant lectins have been reported as diagnostic markers for malignant cells. We studied 51 aspirated samples of benign and malignant effusions using horseradish-peroxidase-conjugated jackfruit lectin. No significant difference was observed between the cells of pleural and peritoneal fluids. The reactively proliferated mesothelial cells of benign effusions showed a predominance of mild staining while moderate and intense staining was predominant in malignant effusions. Intense and irregular lectin binding was observed in macrophages irrespective of the cause of effusion. The lectin staining method therefore appears to have some clinical significance as an additional diagnostic aid for use in effusion cytology.