Béla Kajtár

Automated fluorescent in situ hybridization (FISH) analysis of t(9;22)(q34;q11) in interphase nuclei

For chronic myeloid leukemia, the FISH detection of t(9;22)(q34;q11) in interphase nuclei of peripheral leukocytes is an alternative method to bone marrow karyotyping for monitoring treatment. With automation, several drawbacks of manual analysis may be circumvented. In this article, the capabilities of a commercially available automated image acquisition and analysis system were determined by detecting t(9;22)(q34;q11) in interphase nuclei of peripheral leukocytes.

State-of-the-art FISHing: Automated analysis of cytogenetic aberrations in interphase nuclei

Interphase fluorescence in situ hybridization (i‐FISH) is a powerful tool for visualizing various molecular targets in non‐dividing cells. Manual scoring of i‐FISH signals is a labor intensive, time‐consuming, and error‐prone process liable to subjective interpretation. Automated evaluation of signal patterns provides the opportunity to overcome these difficulties. The first report on automated i‐FISH analysis has been published 20 years ago and since then several applications have been introduced in the fields of oncology, and prenatal and fertility screening. In this article, we provide an insight into the automated i‐FISH analysis including its course, brief history, clinical applications, and advantages and challenges. The lack of guidelines for describing new automated i‐FISH methods hampers the precise comparison of performance of various applications published, thus, we make a proposal for a panel of parameters essential to introduce and standardize new applications and reproduce previously described technologies.

Estrogen-dependent up-regulation of TRPA1 and TRPV1 receptor proteins in the rat endometrium.

Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors expressed predominantly in sensory nerves are activated by inflammatory stimuli and mediate inflammation and pain. Although they have been shown in the human endometrium, their regulation and function are unknown. Therefore, we investigated their estrogen- and progesterone-dependent alterations in the rat endometrium in comparison with the estrogen-regulated inflammatory cytokine macrophage migration inhibitory factor (MIF). Four-week-old (sexually immature) and four-month-old (sexually mature) female rats were treated with the non-selective estrogen receptor (ER) agonist diethylstilboestrol (DES), progesterone and their combination, or ovariectomized. RT-PCR and immunohistochemistry were performed to determine mRNA and protein expression levels respectively. Channel function was investigated with ratiometric [Ca(2+)]i measurement in cultured primary rat endometrial cells. Both TRP receptors and MIF were detected in the endometrium at mRNA and protein levels, and their localizations were similar. Immunostaining was observed in the immature epithelium, while stromal, glandular and epithelial positivity were observed in adults. Functionally active TRP receptor proteins were shown in endometrial cells by activation-induced calcium influx. In adults, Trpa1 and Trpv1 mRNA levels were significantly up-regulated after DES treatment. TRPA1 increased after every treatment, but TRPV1 remained unchanged following the combined treatment and ovariectomy. In immature rats, DES treatment resulted in increased mRNA expression of both channels and elevated TRPV1 immunopositivity. MIF expression changed in parallel with TRPA1/TRPV1 in most cases. DES up-regulated Trpa1, Trpv1 and Mif mRNA levels in endometrial cell cultures, but 17β-oestradiol having ERα-selective potency increased only the expression of Trpv1. We provide the first evidence for TRPA1/TRPV1 expression and their estrogen-induced up-regulation in the rat endometrium in correlation with the MIF.

CD99-positive undifferentiated round cell sarcoma diagnosed on fine needle aspiration cytology, later found to harbour a CIC-DUX4 translocation: a recently described entity

IntroductionPoorly differentiated, small round cell sarcomasoften represent a serious differential diagnostic prob-lem. CD99 expression was initially considered to bespecific for Ewing sarcoma (ES); however, it mayalso be seen in many histologically similar neo-plasms. The differential diagnosis of these tumoursincludes mesenchymal chondrosarcoma, poorly dif-ferentiated synovial sarcoma and lymphoblastic lym-phoma. However, rarely, CD99 expression mayappear in other neoplasms of a similar morphologysuch as the blastemal component of Wilms’ tumour,small cell osteosarcoma, rhabdomyosarcoma, smallcell carcinomas, etc., further expanding the list ofdifferentials.

Automated FISH analysis using dual‐fusion and break‐apart probes on paraffin‐embedded tissue sections

Detecting balanced translocations using tissue sections plays an important diagnostic role in cases of hematological malignancies. Manual scoring is often problematic due to truncation and overlapping of nuclei. Reports have described automated analysis using primarily tile sampling. The aim of this study was to investigate an automated fluorescent in situ hybridization analysis method using grid sampling on tissue sections, and compare the performance of dual‐fusion (DF) and break‐apart (BA) probes in this setting. Ten follicular, 10 mantle cell lymphoma, and 10 translocation‐negative samples were used to set the threshold of false positivity using IGH/CCND1, IGH/BCL‐2 DF, and IGH BA probes. The cut‐off distances of red and green signals to define fusion signals were 0.5, 1.0, and 1.2 μm for the IGH/CCND1, IGH/BCL‐2 DF, and IGH BA probes, respectively. The mean false positivity of grid units was 5.3, 11.4, and 28.1%, respectively. Ten to 14 additional samples analyzed blindly and were correctly classified using each probe. Discriminating positive and negative samples using automated analysis and grid sampling was possible with each probe, although different definitions of fusion signals were required due to the different physical distances between the DNA probes. Using the DF probes resulted in lower false positivity, which was less affected by signal numbers per grid units.

Multiplex Ligation-Dependent Probe Amplification and Fluorescence In Situ Hybridization are Complementary Techniques to Detect Cytogenetic Abnormalities in Multiple Myeloma

Multiple myeloma (MM) is a genetically heterogeneous disease with diverse clinical outcomes. Interphase fluorescence in situ hybridization (i‐FISH) is the most commonly used approach to detect recurrent cytogenetic abnormalities in this malignancy. We aimed to assess the performance of multiplex ligation‐dependent probe amplification (MLPA) to reveal copy number abnormalities (CNAs) in MM. Diagnostic bone marrow samples from 81 patients were analyzed using 42 MLPA probes for the following regions: 1p32‐31, 1p21, 1q21.3, 1q23.3, 5q31.3, 12p13.31, 13q14, 16q12, 16q23, and 17p13. All samples were also screened by i‐FISH for the presence of hyperdiploidy, deletion/monosomy of chromosome 13, deletion of TP53, disruption of the immunoglobulin heavy‐chain gene, t(4;14), t(11;14), t(14;16), t(8;14), gain of 5q and abnormalities of chromosome 1. A total of 245 alterations were detected in 79 cases (98%). Investigating the same aberrations, the two methods showed a congruency of higher than 90%. A low proportion of cells with the relevant abnormality, focal CNAs and unmatched probes were responsible for the discrepancies. MLPA revealed 95 CNAs not detected by i‐FISH providing additional information in 53 cases (65%). Scrutiny of CNAs on chromosome 1, using more than 20 probes, revealed significant heterogeneity in size and location, and variable intra‐chromosomal and intra‐clonal rates of loss or gain. Our results suggest that MLPA is a reliable high‐throughput technique to detect CNAs in MM. Since balanced aberrations are key to prognostic classification of this disease, MLPA and i‐FISH should be applied as complementary techniques in diagnostic pathology.

Urovysion: Considerations on modifying current evaluation scheme, including immunophenotypic targeting and locally set, statistically derived diagnostic criteria

Urovysion multitarget fluorescence in situ hybridization (FISH) assay is a promising tool for detection of bladder cancer, however, there is still no consensus regarding abnormal signal pattern and cut‐off level, and the recommended targeting carries limitations similar to urine cytology. Aim of this study was to explore diagnostic benefits of a recently introduced method featuring target specific genotyping, as well as to investigate the feasibility of locally and statistically determined cut‐off, compared with conventional evaluation scheme. Histology, cytology, and comparative FISH approaches were performed on 42 patients with high clinical suspicion for urothelial carcinoma (UC). FISH parallels were (1) Urovysion‐alone (according to manufacturers instruction); (2) Targeted‐Urovysion (cytokeratin7 immunophenotyping followed by Urovysion), both of which evaluated by both conventional and statistical evaluation scheme. For statistical evaluation cut‐offs and sufficient sample size were determined on controls and ratio of positive cells was recorded, whereas conventional evaluation relied on manufacturers recommendations. The specificity of cytology, Urovysion‐alone in general and targeted‐Urovysion in general appeared 86%, 86%, and 100%, respectively. In the same comparison, overall sensitivity was 60%, 80%, and 93%, respectively. In superficial cases sensitivity was 48% for cytology, 72% for Urovysion‐alone and 91% for targeted‐Urovysion, while no prominent differences were seen in muscle invasive cases. The ratio of FISH positive cells was proportionate with both stage and grade, however, targeted genotyping could separate high grade/high stage cases more effectively. In conclusion, CK7 targeting raises diagnostic efficiency of Urovysion, and could be an ideal tool for identifying tumor cells in ambiguous cases or when other tumors are present. Statistical evaluation produces accuracy comparable with results of conventional evaluation, and with laboratories setting cut‐offs individually but according harmonized protocol, it could aid method standardization. Furthermore, by providing additional quantitative information about tumor characteristics, is likely to have therapy relevant value in the future.

Increased efficiency of detecting genetically aberrant cells by UroVysion test on voided urine specimens using automated immunophenotypical preselection of uroepithelial cells

There is a steady search for procedure which could replace or at least reduce the frequency of the invasive cystoscopy in the surveillance of heterogeneous superficial transitional cell carcinoma (TCC) of the bladder. Recently, UroVysion FISH assay has been shown to provide with better sensitivity than the urine cytology except for the lowest stage pTa and grade I‐II TCCs. Data indicate that this failure of the sensitive FISH might be due to mistargeting. Therefore, our aim was to elaborate a procedure enabling FISH analysis in phenotypically preselected urothelial cells, only. Cytokeratin 7 (CK‐7) chromogenic immunolabeling was applied to various mixtures of negative and positive control cells as well as voided urine specimens. Cellular targets and CK‐7 positive cells were identified by morphometric and pixel intensity indices using an automated microscope workstation. UroVysion FISH pattern was analyzed only in the subsequently relocalized CK‐7 positive events. Automated phenotypical preselection of urothelial cells proved to have 97.3% sensitivity, 96.1% specificity, and 99.0% accuracy, whereas combined pheno‐ and genotyping revealed 93.3% sensitivity and 99.8% specificity, respectively. In clinical samples, the overall 20.4% FISH positivity gained by traditional target identification contrasted with the 55.6% positivity obtained by the combined method, by which the efficiency of identifying chromosomally aberrant cells proved to be two to threefold higher even in grade I lesions. FISH analysis of phenotypically preselected urothelial cells might represent a reliable asset in surveillance of low stage–low grade TCCs.

Automated signal pattern evaluation of a bladder cancer specific multiprobe-fish assay applying a user-trainable workstation.

OBJECTIVE: Signal pattern enumeration of Urovysion Fluorescence in Situ Hybridization test is tedious and requires great experience. Our aim was to eliminate human interaction by automating the process, using an adoptable, automated image acquisition, and analysis system. METHODS: For extensive analytical analysis control, cell populations were used, while preliminary clinical study was performed on 21 patients with clinical suspicion for bladder cancer. All investigations were carried out using an automated user‐trainable workstation (Metafer4‐Metacyte). RESULTS: The system identified nuclei with a specificity and sensitivity of 92.7 and 96.6%, respectively, while signal detection accuracy was 81.1% on average. Both analytical and diagnostic accuracy of automated analysis was comparable to manual approach (94.8 and 71% vs. 97.9 and 76%, respectively), but classification accuracy increased with degree of polysomy, thus diagnostic sensitivity in low grade, low stage cases was poor. CONCLUSION: It is possible to automate signal enumeration of Urovysion using a user‐trainable system, and achieve efficiency comparable to manual analysis. Previously introduced automated immunophenotypic targeting should further increase diagnostic sensitivity, while resulting in a comprehensively automated method. However, the problem of reduced detection accuracy in cases featured with low polysomy is likely to remain a great challenge of automated signal enumeration. Microsc. Res. Tech. 75:814–820, 2012.

MLPA is a powerful tool for detecting lymphoblastic transformation in chronic myeloid leukemia and revealing the clonal origin of relapse in pediatric acute lymphoblastic leukemia

Copy number alterations (CNAs) at 58 different loci have been investigated in 95 bone marrow or peripheral blood samples from patients with chronic myeloid leukemia (CML) or pediatric acute lymphoblastic leukemia (pALL) using multiplex ligation-dependent probe amplification (MLPA). In all but one case, the CNA profile correctly distinguished patients with CML who were in chronic phase from those in lymphoblast crisis. Within the chronic phase group, we could not separate patients resistant to imatinib therapy from those who were good responders. In our investigation of patients with pALL, a panel of MLPA probes broader than ever before was applied. Paired diagnostic and relapse samples from patients with pALL demonstrated clonally related or independent dominant clones, suggesting the presence of a pre-leukemic cell group. Identification of the origin of cell populations dominating at relapse will have a great effect on future treatment strategies. In summary, we have demonstrated the versatility of MLPA by using this cost-effective technique for two new applications.