Managing Difficulties of Microsatellite Instability Testing in Endometrial Cancer-Limitations and Advantages of Four Different PCR-Based Approaches.

Microsatellite instability (MSI), a common alteration in endometrial cancers (EC) is known as a biomarker for immune checkpoint therapy response alongside screening for Lynch Syndrome (LS). However, former studies described challenging MSI profiles in EC hindering analysis by using MSI testing methods intensively validated for colorectal cancer (CRC) only. In order to reduce false negatives, this study examined four different PCR-based approaches for MSI testing using 25 EC samples already tested for mismatch repair deficiency (dMMR). In a follow up validation set of 75 EC samples previously tested both for MMR and MSI, the efficiency of a seven-marker system corresponding to the Idylla system was further analyzed. Both Bethesda and Promega marker panels require trained operators to overcome interpretation complexities caused by either hardly visible additional peaks of one and two nucleotides, or small shifts in microsatellite repeat length. Using parallel sequencing adjustment of bioinformatics is needed. Applying the Idylla MSI assay, an evaluation of input material is more crucial for reliable results and is indispensable. Following MMR deficiency testing as a first-line screening procedure, additional testing with a PCR-based method is necessary if inconclusive staining of immunohistochemistry (IHC) must be clarified.

Detection of HPV subtypes by mass spectrometry in FFPE tissue specimens: a reliable tool for routine diagnostics.

AIMS: Human papilloma virus (HPV) infection is a causative agent for approximately 5% of all new cancer cases in humans. The virus is detected in cervical, anal, vaginal, penile, vulvar and head and neck cancers and has prognostic implications. Thus, test systems are required to detect high-risk but also low-risk HPV subtypes with high specificity and sensitivity in a time-effective and cost-effective manner. In the present study we developed a new mass spectrometry (MS)-based test system for the detection of HPV infections in formalin-fixed paraffin-embedded (FFPE) tissue samples. METHODS: A high-throughput matrix-assisted laser desorption ionisation time of flight MS-based assay was applied to genotype 19 HPV types in FFPE tissue specimens (n=46). The results from the MS assay were compared with the results obtained from two hybridisation-based test systems: the HPV 3.5 LCD-array kit and the EuroArrayHPV system. RESULTS: In 36 out of 46 (78%) tissue samples, a HPV infection could be detected by the MS-based HPV assay. In 16 samples (44%) only one and in 20 samples (56%) two to six HPV subtypes were identified. The overall agreement of all three assays was almost perfect (Cohen's k value: 0.83). CONCLUSIONS: The MS-based assay is highly sensitive, reliable as well as cost-effective and represents a suitable technology for the detection of HPV infections in FFPE tissue material.

Expression of miR-146a, miR-155, and miR-223 in formalin-fixed paraffin-embedded synovial tissues of patients with rheumatoid arthritis and osteoarthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease with a heterogeneous clinical presentation affecting about 1 % of adults in developed countries. Currently, the diagnosis is based on the revised criteria of the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) from 2010. These criteria include clinical and laboratory parameters. Because of the variability of the clinical picture, delayed diagnosis of RA occurs in a significant subset of patients. Therefore, the discovery of novel biomarkers that improve the diagnosis of RA is of particular interest. Recently, it became evident that miRNAs have regulatory activities in physiologic processes and human diseases. Upregulation of miR-146a, miR-155, and miR-223 has been shown in various compartments such as serum, blood, synovial fluid, and tissues in patients with RA. A total of 87 samples were analyzed (RA 50, osteoarthritis (OA) 37). RNA was isolated from formalin-fixed paraffin-embedded synovial tissue (FFPE). The relative expression of miR-146a, miR-155, and miR-223 was determined by comparison to a housekeeping RNA molecule (snRNA U6) and an RNA pool from histologically and clinically verified OA samples. miR-146a, miR-155, and miR-223 were significantly elevated in RA compared to OA synovial tissues (p < 0.001). A strong correlation between the miRNAs could be observed. The sensitivity and specificity for the detection of RA were 0.76/0.80 (miR-146a), 0.80/0.95 (miR-155), and 0.86/0.81 (miR-223). The combination of miR-155 and miR-223 resulted in the highest area under the curve (AUC 0.92) with a sensitivity and specificity of 0.84/0.91, respectively. Significantly higher expression levels of miR-146a, miR-155, and miR-223 in FFPE synovial tissue samples of patients with established RA compared to patients with OA were shown. The usefulness of these miRs for the differential diagnosis of early phases of RA against OA remains to be investigated.

Detection of KRAS, NRAS and BRAF by mass spectrometry - a sensitive, reliable, fast and cost-effective technique.

BACKGROUND: According to current clinical guidelines mutational analysis for KRAS and NRAS is recommended prior to EGFR-directed therapy of colorectal cancer (CRC) in the metastatic setting. Therefore, reliable, fast, sensitive and cost-effective methods for routine tissue based molecular diagnostics are required that allow the assessment of the CRC mutational status in a high throughput fashion. METHODS: We have developed a custom designed assay for routine mass-spectrometric (MS) (MassARRAY, Agena Bioscience) analysis to test the presence/absence of 18 KRAS, 14 NRAS and 4 BRAF mutations. We have applied this assay to 93 samples from patients with CRC and have compared the results with Sanger sequencing and a chip hybridization assay (KRAS LCD-array Kit, Chipron). In cases with discordant results, next-generation sequencing (NGS) was performed. RESULTS: MS detected a KRAS mutation in 46/93 (49%), a NRAS mutation in 2/93 (2%) and a BRAF mutation in 1/93 (1%) of the cases. MS results were in agreement with results obtained by combination of the two other methods in 92 (99%) of 93 cases. In 1/93 (1%) of the cases a G12V mutation has been detected by Sanger sequencing and MS, but not by the chip assay. In this case, NGS has confirmed the G12V mutation in KRAS. CONCLUSIONS: Mutational analysis by MS is a reliable method for routine diagnostic use, which can be easily extended for testing of additional mutations.

The urokinase-system--role of cell proliferation and apoptosis.

The serine protease urokinase-type plasminogen activator (uPA) and its receptor (uPAR) are involved in the control of extracellular matrix turnover, cell migration, invasion and cell signalling leading to a variety of different responses, under both physiological and pathological conditions. The urokinase receptor, binding to the growth factor-like domain of uPA, directs membrane-associated extracellular proteolysis and signals through transmembrane proteins, thus regulating tissue regeneration, angiogenesis, cancer growth and metastasis. Since these physiological and patho-physiological processes of the uPA-system are known, less informations concerning uPA-induced cell proliferation and anti-apoptotic effects of the uPA-system are available. Recent studies show a close relationship of the uPA-system and cell proliferation/ apoptosis. uPA is responsible for the activation and release of different growth factors and modulates the cell proliferation/apoptosis ratio through the dynamic control of cell-matrix interactions. This article focuses on the important role of the uPA/uPAR-system for cell proliferation and apoptosis.

The expression of metastasis suppressor MIM/MTSS1 is regulated by DNA methylation.

MIM/MTSS1 was initially described as a gene missing in invasive bladder cancer cell lines. Functional analysis revealed that MIM is an actin binding protein involved in the regulation of actin cytoskeleton dynamics. MIM was shown to be sonic hedgehog (Shh) signaling dependent and synergizes with the effects of Gli transcription factors. Overexpression of MIM in cell lines leads to the inhibition of cell proliferation. In this study, we showed that the inhibition of cell growth by MIM is anchorage independent. We identified and cloned the promoter region of MIM and located the main promoter activity to 276 bp of 5' flanking sequence sited within a CpG island. Analysis of DNA methylation using bisulphite sequencing revealed that MIM promoter is methylated in its 5' region in cells and tissue samples with reduced endogenous MIM expression. Using luciferase reporter assay, we demonstrated that nonmethylated MIM promoter has a similar activity in cell lines with different endogenous MIM expression. Inhibition of DNA methylation by 5-Aza-2'-deoxycytidine led to upregulation of MIM expression in a low expressing cell line. In conclusion, we clearly demonstrate here that the expression of metastasis suppressor MIM is regulated by DNA methylation of a CpG island within its promoter region.

The coincidence of chromosome 15 aberrations and beta2-microglobulin gene mutations is causative for the total loss of human leukocyte antigen class I expression in melanoma.

PURPOSE: Total loss of surface presentation of human leukocyte antigen (HLA) class I molecules, protecting tumor cells from the recognition by cytotoxic host CD8+ T cells, is known to be caused by mutations in the beta2-microglobulin (beta2m) gene. We asked whether abnormalities of chromosome 15, harboring the beta2m gene on 15q21, in addition to beta2m gene mutations, are causative for the HLA class I-negative phenotype of melanoma cells. EXPERIMENTAL DESIGN: To answer this, we established primary cell lines from the beta2m-negative metastatic melanoma tissues of four different patients and analyzed them for beta2m gene mutations and chromosome 15 aberrations, the latter by loss of heterozygosity analysis, fluorescence in situ hybridization (FISH), and multicolor FISH. RESULTS: Mutations at the beta2m gene level were detected in all cell lines. The loss of heterozygosity analysis of microsatellite markers located on chromosome 15 in three of the four cell lines pointed to an extensive loss of chromosome 15 material. Subsequent molecular cytogenetic analysis revealed the coexistence of apparently normal and rearranged versions of chromosome 15 in three cell lines whereas the fourth cell line solely showed rearranged versions. Two of the four cell lines exhibited a special type of intrachromosomal rearrangement characterized by FISH signals specific for the subtelomeric region of 15q at both ends of the chromosome and one centromeric signal in between. CONCLUSIONS: Our data indicate that the complete loss of HLA class I expression in melanoma cells is due to the coincidence of the following mutational events: (a) chromosome 15 instability associated with an extensive loss of genetic material and (b) beta2m gene mutations.

Urokinase-type plasminogen activator induces proliferation in breast cancer cells.

Urokinase-type plasminogen activator (uPA) is implicated in various pathophysiological processes, including extracellular matrix turnover, cell migration and invasion. Our study aimed to determine the role of uPA in both proliferation and mitogen-activated protein kinase (MAPK) pathway. Hence, we analyzed the effects induced by exogeneous addition of domain-specific uPA antibodies and uPA-interacting molecules on proliferation of uPA-suppressed MDA-MB-231 breast cancer cells. uPA expression was reduced to 53% by stable transfection with an antisense/vector construct and to 65% by siRNA transfection. Immunocytochemical Ki67 staining and flow cytometry (S-phase) analysis indicated a strong decrease of cellular proliferation activity (35% and 38%, respectively). Exogenous addition of high molecular weight-uPA (HMW-uPA) or incubation with the amino terminal fragment (ATF), which lacks the enzymatic activity of uPA, lead to increased cell proliferation. A strong increase of proliferation was absent when the monoclonal anti-uPAR antibody IIIF10 (blocking uPA binding site), soluble uPAR (scavenger effect) and phosphatidyl-inositol-specific phospholipase C (PI-PLC, degrading uPAR) was added prior to the addition of HMW-uPA. In conclusion, HMW-uPA and ATF induce proliferation of breast cancer cells by binding to uPAR. Thereby, integrins situated adjacent to uPAR carry the signals into the cell, thus stimulating proliferation that is mediated via the MAPK pathway.

Dual FISH analysis of benign and malignant tumors of the salivary glands and paranasal sinuses.

To date, the underlying genomic changes in benign and malignant tumors of salivary-gland and paranasal-sinus origin are poorly understood. This is due in part to the low incidence of these tumors and the enormous histological variety of tumors within this head and neck region. We examined 58 of these tumors (14 adenoid cystic carcinomas, 9 adenocarcinomas, 5 cylindrical carcinomas, 11 pleomorphic adenomas, and 19 inverted papillomas) by dual fluorescence in situ hybridization (FISH) with centromere-specific probes on six chromosomes (3, 7, 9, 11, 17, and 18) for numerical changes. In adenoid cystic carcinomas, monosomy of chromosome 17 and polysomy of chromosomes 3, 9 and 11 were most frequently encountered. In adenocarcinomas, monosomy of chromosome 17 and polysomy of chromosomes 7 and 11 were most frequent. In cylindrical cell carcinomas, polysomy of chromosomes 7, 9, 11 and 17 was present in the majority of tumors. Disomy is rare, even in benign tumors. Polysomy is more frequent in malignant tumors than in benign. Tetrasomy is found almost only in malignant tumors. In summary, the occurrence of polysomy might reflect a step towards malignancy in tumors of the salivary glands and paranasal mucosa. Polysomy of chromosome 11 could be defined as typical for all investigated histological types of malignant tumor in this region of the head and neck.

HER2/neu, p53, Ki67, and hormone receptors do not change during neoadjuvant chemotherapy in breast cancer.

The selection of a systemic breast cancer therapy is based on the expression pattern of immunohistochemical prognostic markers. In our study we sought to determine whether neoadjuvant chemotherapy may alter these expression patterns within the tumors. Our hypothesis was that the expression of the immunohistochemical prognostic markers does not differ between tissue specimens before and after neoadjuvant chemotherapy. We determined the protein expression levels of estrogen receptor, progesterone receptor, Ki67, p53 and HER2/neu in the core biopsy and the resected tumor sample from 25 patients receiving neoadjuvant chemotherapy. As a control group, we analyzed sample pairs from 30 patients who did not receive neoadjuvant chemotherapy. Additionally, we determined the relative HER2/neu gene copy number by FISH and/or real-time PCR. There were no significant differences in the changes in expression patterns from the core biopsy to the treated resected tumor between those who had received neoadjuvant chemotherapy and the control group. We suggest that it is sufficient to analyze the prognostic factors from either the core biopsy prior to chemotherapy or the treated tumor sample instead of investigating both samples. This would markedly reduce the costs.

In vitro suppression of urokinase plasminogen activator in breast cancer cells--a comparison of two antisense strategies.

High level expression of urokinase plasminogen activator (uPA) has been well documented in a variety of tumors. In breast cancer, expression of uPA is essential for tumor cell invasion, metastasis and proliferation. By contrast, the primary objective of tumor therapy is to reduce the uPA expression level within the tumor, which results in abrogation of proliferation, invasion and metastasizing of the tumor cells. We investigated the effects of uPA on the MDA-MB-231 cell line. MDA-MB-231 cells are highly invasive and express high levels of uPA. In our study, uPA inhibition was achieved by two methodologies: a) stable transfection with an antisense uPA vector, b) transfection with siRNA molecules (small interfering RNA). A plasmid vector was constructed by cloning a uPA-specific cDNA (612 bp) fragment into pBK-CMV plasmid in antisense orientation. In contrast, a double-stranded 21-mer siRNA was designed for targeting uPA. The antisense-transfected cells revealed decreased uPA mRNA and protein as detected by real-time PCR, immunocytochemistry, ELISA, and Western blotting. Moreover, the transfected cells exhibited a significantly reduced proliferation activity as determined by a fluorometric proliferation assay. As a conclusion of our study siRNA-technique is the superior method also regarding time saving for clone selection and instant availability of the transfected cells. Moreover, even if both strategies lead to uPA suppression, a stronger inhibitory effect could be obtained by application of the siRNA-based technique.

Intratumoral genomic heterogeneity in primary head and neck cancer and corresponding metastases detected by dual-FISH.

Intratumoral genomic heterogeneity, which can be defined as both intersample and intrasample heterogeneity, is still a poorly understood phenomenon in head and neck squamous cell carcinoma (HNSCC) with presumed implications on tumor behavior and even prognosis. We analyzed 89 tumor specimen from 37 HNSCC patients by fluorescence in situ hybridization (dual-FISH) using specific DNA probes binding to centromeric sites of 6 chromosomes to investigate intratumoral heterogeneity. A derivation from disomy in at least 1/6 chromosomes was detected in 88/89 (99%) specimen. In 33% of these samples, a change in ploidy could be suspected. Intrasample heterogeneity was detected in 68/89 (76%). Intrasample heterogeneity was more pronounced in primary tumors than in metastatic tumors. Analysis of the intersample heterogeneity revealed notable differences between the 6 chromosomes with the highest discordance detected for chromosome 3 (46%) and the lowest for chromosome 11 (27%). Following our results, it seems important to us to underline that intratumoral heterogeneity exists as intra- and sample heterogeneity in HNSCC. Altogether, trisomic cells were significantly more frequent in primary tumors than in metastases (p=0.01) while, in turn, monosomic cells were significantly more frequent in metastases (p=0.029). In individual cases the extent of discordance between corresponding samples made a common clonal precursor unlikely. In these cases, the synchronous development of a primary tumor and a carcinoma of unknown primary ('CUP syndrome'), otherwise undetected, should be considered.