Centromeric Probes For Chromosomes Research Articles

NEXT-GENERATION GENOMIC MONITORING REVEAL A NOVEL RUNX1 PATHOGENIC MUTATION ASSOCIATED WITH TRISOMY 4 AS A SOLE ABNORMALITY IN A YOUNG ADULT WITH ACUTE MYELOID LEUKEMIA

Acute Myeloid Leukemia (AML) with a single trisomy show particular clinical, morphological, genomic, and immunophenotypic features. Trisomy 4 was first described in 1986 as the sole chromosomal abnormality in AML. However, due to its rarity (< 1%), there are not enough data concerning its clinical impact. Here we report clinical, cyto-molecular, and genomic data of a young adult with AML and a rare trisomy 4 associated with a novel RUNX1 pathogenic mutation. A 19-year-old man was referred to Hospital dos Servidores do Estado, Rio de Janeiro, Brazil. He presented a history of 30 days of anemia, fever, weight loss, thrombocytopenia, and mucocutaneous bleeding. Physical examination revealed hepatomegaly (15 cm) and splenomegaly (14 cm). Laboratory data included hemoglobin level of 6.9 g/dL, WBC of 350 × 10 9 /L, and platelet count of 8 × 10 9 /L. The LDH was 1650 U/L. Flow cytometry analysis revealed 83.4% of blasts showing myeloid morphology that expressed CD34, CD117, CD13, CD7, CD38, and HLA-DR, compatible with FAB AML-M0 with minimal differentiation/undifferentiated. The patient was treated under a high-risk AML protocol. On day 14 of treatment, he developed visceral septicemia and died due to septic shock. We performed G-banding and FISH experiments. NGS was performed on a targeted approach. G-Banding revealed the karyotype 47,XY,+4. FISH with a centromeric probe for chromosome 4 confirmed trisomy 4. Molecular biology tests were positive for FLT3/ITD and negative for NPM1 mutations. NGS results revealed a novel RUNX1 missense pathogenic mutation. Trisomy 4 is a rare chromosomal abnormality in AML, accounting for 1 < % of cases. Although its prognostic relevance has been a frequent topic of discussion in the literature, its relation to prognosis remains to be elucidated. Chilton and coworkers (2016) described 35 patients with trisomy 4 as an isolated abnormality and observed only two patients (6%) in the 16‒25 age group. Patients with trisomy 4 have a poor prognosis when associated with mutations in KIT, mapped at position 4q12, which may influence the role of trisomy 4 in leukemogenesis. The patient in the present study belongs to the same age group as the patients in the abovementioned study. However, to our knowledge, it is the first case with trisomy 4 as a sole abnormality presenting FAB AML-M0. Our patient had a poor prognosis, including hyperleukocytosis. In addition, we characterized a missense RUNX1 mutation not yet described in the literature. RUNX1 is a transcription factor part of a complex that provides stability to the RUNX1 protein, which plays a pivotal role in generating hematopoietic stem cells and their differentiation. Loss of RUNX1 function has been shown to impair cell differentiation, often resulting in leukemogenesis. Besides, RUNX1 mutations are known to cause thrombocytopenia and familial platelet disorder. Here we present a rare AML-M0 case with trisomy 4 as a sole abnormality and highlight its association with a novel RUNX1 mutation. Although, the prognostic implications remain to be elucidated. In particular, when to consider stem cell transplantation as curative therapy of choice in RUNX1 mutated individuals to maximize benefit and minimize risk.

Fluorescence in situ hybridization of thyroid fine-needle aspiration biopsy distinguishes between neoplastic and non-neoplastic Hürthle cell lesions

Fine-needle aspiration (FNA) biopsy of Hürthle cell proliferations can be difficult to characterize based purely on morphologic features. Studies have shown Hürthle cell neoplasms often demonstrate gains in chromosomes 5, 7, and 12. This study examined fluorescence in situ hybridization (FISH) performance characteristics in non-neoplastic and neoplastic Hürthle cell proliferations sampled by FNA biopsy in order to assess chromosome patterns. FNA biopsies of Hürthle cell proliferations, including nodular hyperplasia (NH), Hürthle cell adenoma (HCA), and Hürthle cell carcinoma (HCC), that had subsequent surgical excision were selected. FISH was performed on an air-dried, modified Wright-Giemsa-stained, aspirate smear slide from each case using a 3-color panel consisting of 1 subtelomeric and 2 centromeric probes for chromosomes 5, 7, and 12. Chromosomal probe patterns were recorded in up to 50 cells. A positive result was considered when >15% of cells showed a polysomy in 2 or more chromosomes. A total of 25 cases were included in the study. All cases of NH were negative, and 7 of 9 (78%) HCAs and 8 of 12 (67%) HCCs were positive. Of the positive cases, 2 of the 7 (29%) HCAs showed >50% of cells with polysomy, and 5 of the 8 (63%) HCCs showed >50% of the cells with polysomy. Thyroid FNA biopsy can identify Hürthle cell proliferations; risk stratification based on morphology is difficult, however. FISH chromosomal evaluation of thyroid FNA biopsies is useful to distinguish neoplastic from non-neoplastic Hürthle cell proliferation.

Molecular features of metanephric adenoma and their values in differential diagnosis

Objective: To study the molecular features of metanephric adenoma (MA) and discuss their values in differential diagnosis. Methods: BRAF V600E immunohistochemistry (IHC) using the mutation-specific VE1 monoclonal antibody and Sanger sequencing of BRAF mutations were performed on 21 MAs, 16 epithelial-predominant Wilms tumors (e-WT) and 20 the solid variant of papillary renal cell carcinomas (s-PRCC) respectively. p16 protein was detected by IHC also. Fluorescence in situ hybridization (FISH) analyses using centromeric probes for chromosome 7 and 17 were performed on the three renal tumors in parallel. Results: Fourteen (14/21, 66.7%) of 21 MA cases demonstrated diffuse, moderate to strong cytoplasmic BRAF V600E IHC staining and the BRAF V600E protein expression was detected in 2 (2/16) of 16 e-WT cases for the first time, whereas all s-PRCCs were negative (P<0.05). All cases (including 14 MAs and 2 e-WTs) with diffuse, moderate to strong cytoplasmic BRAF V600E IHC staining were confirmed to harbor BRAF V600E missense mutations using Sanger sequencing, and no BRAF mutations were detected in cases with negative BRAF V600E protein expression. One case (1/21, 4.8%) showed trisomy of chromosome 7 alone, and another one (1/21, 4.8%) showed trisomy of chromosome 17 alone in 21 MAs. Two cases (2/16) of 16 e-WTs showed trisomy of chromosome 17 alone. In 20 s-PRCCs, trisomy of chromosomes 7 alone was reported in 2 cases (2/20), trisomy of chromosome 17 alone in 3 cases (3/20) and trisomy of chromosome 7 and 17 in 14 cases (14/20). The total positive rates of trisomy of chromosome 7 and/or 17 in MAs, e-WTs and s-PRCCs were 9.6% (2/21), 2/16 and 95.0% (19/20). p16 protein was positive in 81.0% (17/21) MAs, whereas the positive rates in e-WTs and s-PRCCs were 2/16 and 5.0% (1/20). Conclusions: Most MAs harbor BRAF V600E mutations, and MAs lack the gains of chromosome 7 and 17 that are characteristic of papillary renal cell carcinoma. These molecular features can be used to distinguish MA from its mimics. BRAF V600E IHC using the mutation-specific VE1 monoclonal antibody provides an effective method in BRAF V600E mutations detection of renal tumors. p16 is overexpressed in MA, and the finding suggests that the low proliferative rate of the tumor might be attributed to BRAF V600E-induced senescence mediated by p16.

Influence of CRBN, IKZF1, and IKZF3 expression on outcome in lenalidomide treated newly diagnosed multiple myeloma patients

RB1,TP53, t(4;14), t(11;14), t(14;16) and a centromeric probes for chromosomes 7, 9, 11, 15 and 17 (Abbott-Vysis Inc., Downers Grove, IL, USA; Dako, Glostrup, Denmark; Kreatech, Amsterdam, The Netherlands; MetaSystems, Altlusheim, Germany). Some complex chromosomal rearrangements were analyzed by using mFISH and arrayCGH methods (MetaSystems;Agilent, Santa Clara,CA,USA). Results: Patients with the absence of delRB1 had significantly increased levels of sOPG (p 1⁄4 0.025) and sVEGF (p 1⁄4 0.036). The presence of t(11;14) was associated with lower levels of sIL-6R (p 1⁄4 0.009). The presence of t(14;16) was associated with higher levels of 2m (p 1⁄4 0.022), and lower serum levels of DKK-1 (p 1⁄4 0.023). The presence of gain 1q21 was associated with higher levels of sTK (p 1⁄4 0.026), sIL-6R (p 1⁄4 0.01), and lower serum levels of PINP (p 1⁄4 0.030). Conclusion: This showed that some cytogenetic changes have an impact on individual components of the cytokine network in MM. The presence of adverse cytogenetic changes is associated with higher levels of neoplastic mediators (sTK, sIL-6R, 2m) and a decrease in levels of bone formation parameters (sOPG, PINP and DKK-1). Identified data complete the image of biology of neoplastic processes in MM. Supported by IGA CR NT 12451/5, NT 14400, NT 14393

Oral lichen planus patients exhibit consistent chromosomal numerical aberrations: A follow-up analysis.

Oral lichen planus (OLP) carries an increased risk for malignant transformation with aneuploid cells (ACs) being found in brush samples of a quarter of patients with OLP. Patients with OLP were followed and repeated brush samples were simultaneously analyzed for morphology and fluorescent in situ hybridization (FISH) using centromeric probes for chromosomes 2 and 8. Three patients with a high proportion of ACs developed oral cancer. Fifteen patients had ≥1% ACs (13 in affected sites and 2 in nonaffected sites), whereas only 2 of the 15 patients with <1% ACs in the first sample had ≥1% ACs in the second sample. A strong positive correlation between the results of the initial and repeated samples was found. High proportion of ACs in brush samples from patients with OLP may imply an impending malignant transformation. As FISH analysis is consistent over time, it can be used to identify a subgroup of patients who would require close follow-up. © 2015 Wiley Periodicals, Inc. Head Neck 38: E741-E746, 2016.

ІНФОРМАЦІЙНО-СТАТИСТИЧНІ ОЦІНКИ ПРИ ЗАСТОСУВАННІ FISH-МЕТОДУ ДЛЯ ВИЯВЛЕННЯ "ПРИХОВАНОГО" МОЗАЦИЗМУ СТАТЕВИХ ХРОМОСОМ СЕРЕД БЕЗПЛІДНИХ ПОДРУЖНІХ ПАР

The application of FISH-method on interphase cells with centromeric probes for chromosomes X and Y is described as used together with the traditional cytogenetic methods for the screening minor sex aneuploid cell lines among the couples with reproductive problems.

Diagnóstico de aneuplodias en células en interfase de líquido amniótico mediante hibridación in situ fluorescente. Concordancia con el cariotipo

BackgroundThe application of fluorescence in situ hybridization (FISH) techniques in the last few years has led to the prenatal diagnosis of aneuploidies. The objective of this investigation was a descriptive analysis of amniotic fluids processed in the laboratory using FISH and the agreement with the karyotype. Material and methodsA total of 821 amniotic fluid samples (January 2009 to December 2010) at gestational ages 13 to 36 weeks, from Fetal Medicine Unit for prenatal testing for aneuploidies (Aneuvysion kit) with centromeric probes for chromosomes X,Y and 18, and locus specific for chromosomes 13 and 21. The study was complemented by the karyotype by G-banding method. ResultsOf the 821 samples, 776 (94.52%) were normal and 45 (5.48%) had aneuploidy: in 22 cases (48.88%) the chromosomal sex of the foetus was male, in the remaining 23 (51.12%) female. The most common chromosomal abnormality detected was trisomy 21 (19 cases in males and 11 cases in female foetuses), the lowest performance was the trisomy 13 (2 cases) representing 0.24% of total processed amniotic fluids. In all cases, the concordance with the karyotype was 100%. ConclusionsThe study of aneuploidy by FISH of interphase nuclei present in the amniotic fluid enables rapid prenatal diagnosis of major chromosomal abnormalities, trisomy 21 was more frequently detected. FISH results in 100% agreed with the karyotype, the gold standard in prenatal diagnosis of chromosomal abnormalities

Polysomy 8 in Egyptian patients with de-novo acute myeloid leukemia

Background Tetrasomy, pentasomy, and hexasomy of the entire chromosome 8 (polysomy 8) are relatively rare compared with trisomy 8, which is one of the most common recurring aberrations in myeloid hematologic malignancies. Although polysomy 8 appears to adversely influence outcome, little is known about the prognostic significance of polysomy 8 in patients with acute myeloid leukemia (AML). Aim of the work The aim of the present work was to study the numerical aberrations of chromosome 8 in Egyptian patients with AML and to clarify its role as a prognostic marker. Patients and methods The present study was carried out on 76 newly diagnosed adult patients with AML. They were attending the Hematology Department of Ain Shams University Hospitals. They were subjected to conventional cytogenetic analysis using G-banding and fluorescence in-situ hybridization using a fluorophore-labeled centromeric probe for chromosome 8. Results Metaphase and interphase fluorescence in-situ hybridization for detection of polysomy 8 was successfully performed on 72 samples. Patients were divided into two groups: group A comprised patients associated with polysomy 8 (20 patients; 27.8%) and group B comprised patients without polysomy 8 (52 patients; 72.2%). Group A patients had a statistically significant younger age compared with group B (P=0.04). A statistically significant association was seen between polysomy 8 status and hepatosplenomegaly, fever, and lymph node infiltration (P=0.03, 0.05). The presence of hepatosplenomegaly, skin infiltration, lymph node infiltration, and French–American–British subtype was shown to have a significant impact on patient outcome (P=0.05, 0.03, 0.01, and 0.04, respectively). Patients with skin infiltration, CD34 expression, HLA-DR expression, high/intermediate cytogenetic risk, and presence of polysomy 8 had significantly shorter overall survival. Conclusion and recommendations From the current study we conclude that polysomy 8 occurs in 27.8% of de-novo AML cases; 35% of these cases showed tetrasomy of chromosome 8. Polysomy of chromosome 8 was associated with shortened overall survival. Further studies with a larger number of patients are recommended for proper assessment of polysomy 8, especially tetrasomy 8, among Egyptian patients with AML.

Gastric schwannoma: a clinicopathologic study of 51 cases and critical review of the literature

Schwannoma is a rare gastrointestinal mesenchymal tumor, as the vast majority of gastric mesenchymal tumors are gastrointestinal stromal tumors. In this study, we analyzed clinicopathologically 51 gastric schwannomas. These tumors predominantly occurred in older adults with a marked female predominance (40 women and 11 men; median and mean ages, 60 and 58 years). They variably presented with gastric discomfort, bleeding, or rarely gastric outlet obstruction; and many were incidental findings during other medical procedures. The tumors ranged from 1 to 10.5 cm (median, 4.5 cm). The typical histologic features included spindle cells usually with microtrabecular architecture and focal nuclear atypia, and peritumoral lymphoid cuff, whereas features of soft tissue schwannomas, such as encapsulation, nuclear palisading, vascular hyalinization, and dilatation, were absent or infrequent. Median mitotic count was 2/50 high-power fields, with the highest count being 13/50 high-power fields. No malignant variants were recognized, and long-term follow-up did not reveal recurrences or metastases. Immunohistochemically, all examined tumors were S100 protein positive and most were also GFAP positive, whereas CD34 and NF68 were encountered rarely and all tumors were negative for HMB45, KIT, DOG1/Ano 1, smooth muscle actin, desmin, and synaptophysin. None of the 9 tumors studied contained gastrointestinal stromal tumor-specific KIT or PDGFRA mutations. Fluorescence in situ hybridization studies revealed multiple signals with BCR probe (chromosome 22) and centromeric probes for chromosomes 2 and 18 suggesting polyploidy. These findings indicate that gastric schwannoma is a distinctive form of peripheral nerve sheath tumor that in many ways differs from soft tissue schwannoma. It should be distinguished from gastrointestinal stromal tumor and other mesenchymal tumors of the gastrointestinal tract, such as the S100 protein-positive gastrointestinal clear cell sarcoma and metastatic melanoma.

Computerized cell-scanning system for evaluating human spermatogenesis in non-obstructive azoospermic patients

There may be incompatibility between testicular histopathological evaluation and testicular sperm extraction (TESE) outcome. Assessment for sperm presence and different pathological disturbances of non-obstructive azoospermia (NOA) remains challenging. An assay for maximal sampling and accurate identification of testicular cells from NOA patients undergoing TESE and autopsied fertile controls was developed. Testicular cells stained and scanned automatically for morphology underwent fluorescence in-situ hybridization using centromeric probes for chromosomes X, Y and 18 after destaining. Cells were automatically classified according to ploidy, and ratios of haploid cells and autosomal (18) and sex-chromosome bivalent rates were calculated. Identification of testicular cells in suspension enabled prediction of spermatogenesis in seven of eight Sertoli-cell-only syndrome patients. Haploid/diploid cell ratios were 67.6:32.2 for controls and 9.6:90.4 for patients. Both autosomal (18) and sex-chromosome bivalents were present in patients (4.1 ± 5.82%) and controls (19.7 ± 8.95%). Few tetraploid pachytene spermatocytes were observed. More secondary spermatocytes with NOA showed two distinct signals for chromosome 18 (27.9 ± 32.69%) compared with controls (0.4 ± 0.35%). The computerized cell-scanning system enables simultaneous application of morphology and chromosome analysis of testicular cells, which enhance assessing different pathological disturbances and estimating the likelihood of a successful second TESE procedure. Assessment for sperm presence and different pathological disturbances of non-obstructive azoospermia (NOA) remains challenging. There may be incompatibility between testicular histopathological evaluation and testicular sperm extraction outcome. Precise identification of individual germ cells in a wet cell preparation during TESE could contribute to understanding the pathology. An assay for maximal sampling and accurate identification of testicular cells from NOA patients undergoing TESE was developed. A prospective cohort study was conducted at a university-based IVF centre. Cells from the testicular suspension of 12 NOA patients and three autopsied fertile controls were fixed on glass slides using a Cytospin centrifuge and stained with May–Grünwald Giemsa. The slides were automatically scanned for cell morphology. Fluorescence in-situ hybridization using centromeric probes for chromosomes X, Y and 18 was performed after destaining, and cells were automatically classified according to ploidy. Identification of different testicular cells in the suspension enabled prediction of spermatogenesis in seven out of eight Sertoli-cell-only syndrome patients. Ratios of haploid to diploid cells were 67.6:32.2 in the controls and 9.6:90.4 in the study group. Presence of both autosomal (18) and sex chromosome bivalents was 19.7 ± 8.95% in the controls and 4.1 ± 5.82% in the study group ( P < 0.05). Some tetraploid pachytene spermatocytes were identified in both groups. More secondary spermatocytes in the study group showed two distinct signals for chromosome 18 than in controls: 27.9 ± 32.69% versus 0.4 ± 0.35 ( P < 0.02). Thus, computerized cell scanning enables simultaneous application of morphology and chromosomal analysis of testicular cells and examination of meiotic pairing for enhanced assessment of pathological disturbances.

Abstract 3822: DNA copy number changes in the PI3K signaling pathway genes in triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is associated with poor prognosis and a high mortality rate when compared to the other breast cancer subtypes. These tumors lack the expression of ER, PR and HER2 receptors, and do not respond well to the currently available adjuvant therapies. The phosphatidylinositol 3-kinase (PIK3) signaling pathway mediates key cellular functions. Alterations of genes in this pathway have been shown to predict responsiveness to cancer targeted therapies. In this study, five genes involved in this signaling pathway, PIK3CA, AKT1, AKT2, PTEN and FGFR, were evaluated for DNA copy number changes in two sets of TNBC specimens. Methods: TNBC samples were obtained from the Hospital Nossa Senhora das Gracas, Curitiba, PR, Brazil. Two sets of TNBC samples were analyzed: the first set was composed of 30 formalin fixed-paraffin embedded tissue specimens and were analyzed for the PIK3CA, AKT1, AKT2, PTEN and FGFR genes by FISH. Locus-specific probes were prepared from BAC contigs for each gene and a centromeric probe for chromosome 3 was used as a control for copy number. The second set was composed of 17 frozen samples and were analyzed by array-CGH using a 44K-Agilent array platform. Real-Time PCR (TaqMan assay), was used to validate the array-CGH data for the above five genes. Results: FISH: PTEN loss and FGFR3 amplification were the most common findings occurring in 56% and 41% of the TNBC samples respectively. PIK3CA, AKT2 and AKT1 were amplified in 31%, 25% and 9.5% of the samples respectively. Array-CGH/Real time PCR: DNA copy number changes were observed by array-CGH in all, but one TNBC sample. The gene with highest frequency of amplification detected by array-CGH was PIK3CA (amplified in 77% of the cases). These findings were confirmed in 70% of the cases. FGFR2, AKT2 and PTEN were amplified in 35%, 24% and 24% of the cases respectively, These findings were confirmed for FGFR2 in 83% of the samples and for PTEN in all the samples. AKT2 amplification was only confirmed in 25% of the samples. Finally AKT1 was observed amplified in by array-CGH in 6% of the cases, which was confirmed by Real-time PCR.Conclusions: Based on our initial results, we conclude that TNBC presents DNA copy number alterations in genes involved in the PIK3 signaling pathway. Upon validation of these alterations in a larger number of cases, with correlation to prognostic parameters, their potential use as therapeutic targets can be considered for further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3822. doi:10.1158/1538-7445.AM2011-3822

526 ESTABLISHMENT OF AN INTERPHASE FISH TEST FOR PREDICTION OF PROGNOSIS IN CLEAR CELL RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Evaluation and Staging1 Apr 2011526 ESTABLISHMENT OF AN INTERPHASE FISH TEST FOR PREDICTION OF PROGNOSIS IN CLEAR CELL RENAL CELL CARCINOMA Jimsgene Sanjmyatav, Martin Mühr, Doriana Sava, Sophie Matthes, Maria Sternal, Heiko Wunderlich, Thomas Steiner, Marc-Oliver Grimm, and Kerstin Junker Jimsgene SanjmyatavJimsgene Sanjmyatav Jena, Germany More articles by this author , Martin MührMartin Mühr Jena, Germany More articles by this author , Doriana SavaDoriana Sava Jena, Germany More articles by this author , Sophie MatthesSophie Matthes Jena, Germany More articles by this author , Maria SternalMaria Sternal Jena, Germany More articles by this author , Heiko WunderlichHeiko Wunderlich Jena, Germany More articles by this author , Thomas SteinerThomas Steiner Jena, Germany More articles by this author , Marc-Oliver GrimmMarc-Oliver Grimm Jena, Germany More articles by this author , and Kerstin JunkerKerstin Junker Jena, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1250AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES By precise mapping of genomic imbalances of 56 clear cell renal cell carcinomas using array-CGH in our previous studies we identified specific chromosomal aberrations which are significantly associated both with metastasis and cancer specific survival. In multivariate analysis gains of 1q21.3 and of 20q11.21–q13.2 as well as loss of 9p21.3–p24.1 were independent predictors for metastasis. Multivariate Cox-regression analysis revealed gains of 7q36.3 and of 20q11.21–q13.32 and a loss of 9p21.3–p24.1 as independent prognostic factors for outcome of patients. The aim of this study was to prove the prognostic value of these aberrations using FISH probes on these critical regions in order to develop a routine diagnostic tool. METHODS FISH experiments were performed on isolated cell nuclei from tumor tissues. The same cohort of previous array-CGH analysis was used for these experiments (32 metastasized and 24 non-metastasized). For each critical chromosomal region (1q21.3, 7q36.3, 9p21.3–p24.1 und 20q11.21–q13.32) we hybridized a mixture of three different commercially available FISH probes: region specific probe in red dye, centromeric probe of corresponding chromosome or probe close to centromere in green dye and centromeric probe of chromosome 2 in blue colour as control. 100 nuclei were scored in each case. RESULTS Loss of 9p21.3–p24.1 (Fisher's exact test; p=0.017) and gains of 1q21.3 (p=0.030), 7q36.3(p=0.019) and 20q11.21–q13.32 (p=0.008) displayed significant correlations with metastasis occurrence. Our analysis revealed significant correlation of gains 7q36.3 (p=0.001) and 20q11.21–q13.32 (p=0.05) as well as loss of 9p21.3-p24.1 (p=0.00002) with cancer specific death. Patients with tumours harbouring these aberrations in more than 30% of cells have a poor outcom. CONCLUSIONS FISH results confirmed our previous findings of array-CGH and showed that these regions play an important role in cancer progression. Based on these genetic targets it seems possible to design a combined FISH assay which can be used in routine diagnostics for outcome prediction of patients with ccRCC. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e214 Peer Review Report Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jimsgene Sanjmyatav Jena, Germany More articles by this author Martin Mühr Jena, Germany More articles by this author Doriana Sava Jena, Germany More articles by this author Sophie Matthes Jena, Germany More articles by this author Maria Sternal Jena, Germany More articles by this author Heiko Wunderlich Jena, Germany More articles by this author Thomas Steiner Jena, Germany More articles by this author Marc-Oliver Grimm Jena, Germany More articles by this author Kerstin Junker Jena, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

534 CANCER-SPECIFIC SURVIVAL NOMOGRAM FOR RENAL TUMORS WITH VENOUS EXTENSION:INTERNATIONAL RENAL CELL-CARCINOMA-VENOUS THROMBUS CONSORTIUM

benign oncocytomas. As reference to FISH analysis we carried out array-CGH for all analyzed tumors. RESULTS: The correlation of FISH findings with histopathological data revealed that histological subtypes were correct in 100% (26/26) of ccRCC cases, in 80% (8/10) of pRCC, 100% (13/13) in chRCC and in 33% (4/12) of oncocytomas. Two tumors classified as papillary according to histopathological features were grouped to ccRCC on the basis of genetic pattern detected by FISH and CGH analysis. In 8 oncocytomas we found either no genomic alterations or alterations in less than 10% of scored nuclei. Array-CGH and CGH analysis demonstrated no alterations or atypical chromosomal changes in these tumors. CONCLUSIONS: The current study demonstrates that the developed multicolor FISH assay allows an accurate identification of the most frequent renal cell tumor subtypes and increases the diagnostic accuracy of RCC. It represents a robust and rapid method for routine diagnostics in cases of uncertain diagnosis by histopathological evaluation and helps to overcome the limitations of pathological classification. Currently we are working on a second set of probe composed of centromeric probes of chromosomes 2 und 6 and a translocation probe for chromosome 11 allowing the precise differentiation of some ambiguous cases of chromophobe RCC and oncocytomas as well.

Cytogenetics and prognosis for uveal melanoma in Korean patients

To determine the rate of mortality for uveal melanoma in the Korean population and assess whether it correlates with cytogenetic data, and clinical and histopathological factors. A retrospective review of medical files from 33 uveal melanoma patients who underwent enucleation at Yonsei University, Seoul, Korea, between January, 1994 and December, 2009 was performed. Dual-colour fluorescence in situ hybridization was performed using centromeric probes for chromosome 3 and 8 in archived patient tissues. The mean age of patients was 53.2 years (range; 29-78), and the mean largest basal diameter was 11.8 mm (range; 6.0-18.1). Of the 20 tissue blocks with available cytogenetic data, there was monosomy 3 in seven (35%) and polysomy 8 in four (20%). The presence of monosomy 3 (p < 0.001), polysomy 8 (p < 0.001), ciliary body involvement (p < 0.001), a mitotic rate ≥5/40 high-power fields (p = 0.006), closed extravascular matrix loop (p = 0.025), large basal tumour diameter (p = 0.029) and epithelioid cellularity (p = 0.038) predicted melanoma-related mortality. The median time from enucleation to liver metastasis was 16 months (range; 10-70), and the median survival after metastasis was 5 months (range; 1-9). The Kaplan--Meier survival curve estimated the metastatic death rate to be 22% for 5 years and 30% for 10 years. Korean patients with uveal melanoma are younger and appear to exhibit no worse prognosis than Caucasian. Cytogenetic abnormality of chromosome 3 and 8 highly predicted metastatic death.

Clonal heterogeneity and chromosomal instability at disease presentation in high hyperdiploid acute lymphoblastic leukemia

Although aneuploidy has many possible causes, it often results from underlying chromosomal instability (CIN) leading to an unstable karyotype with cell-to-cell variation and multiple subclones. To test for the presence of CIN in high hyperdiploid acute lymphoblastic leukemia (HeH ALL) at diagnosis, we investigated 20 patients (10 HeH ALL and 10 non-HeH ALL), using automated four-color interphase fluorescence in situ hybridization (I-FISH) with centromeric probes for chromosomes 4, 6, 10, and 17. In HeH ALL, the proportion of abnormal cells ranged from 36.3% to 92.4%, and a variety of aneuploid populations were identified. Compared with conventional cytogenetics, I-FISH revealed numerous additional clones, some of them very small. To investigate the nature and origin of this clonal heterogeneity, we determined average numerical CIN values for all four chromosomes together and for each chromosome and patient group. The CIN values in HeH ALL were relatively high (range, 22.2–44.7%), compared with those in non-HeH ALL (3.2–6.4%), thus accounting for the presence of numerical CIN in HeH ALL at diagnosis. We conclude that numerical CIN may be at the origin of the high level of clonal heterogeneity revealed by I-FISH in HeH ALL at presentation, which would corroborate the potential role of CIN in tumor pathogenesis.

Clear Cell Tubulopapillary Renal Cell Carcinoma: A Study of 36 Distinctive Low-grade Epithelial Tumors of the Kidney

Recently several low-grade renal cell tumors, distinct from those recognized by the 2004 World Health Organization classification of renal tumors, have been described. These tumors had similar clinicopathologic features, being low-stage tumors with cystic, tubuloacinar, and/or papillary architecture. The tumor cells were low grade with variable amounts of clear cytoplasm that was positive for cytokeratin 7 (CK7), but negative for CD10. Genetic changes characteristic of clear cell or papillary renal cell carcinoma were not seen in these tumors. We investigated the morphologic, immunohistochemical, and genetic features of 36 additional tumors. Immunohistochemistry was carried out for CK7, carbonic anhydrase 9, α-methylacyl-CoA racemase, CD10, TFE-3, and desmin. Interphase fluorescence in situ hybridization was carried out with centromeric probes for chromosomes 3, 7, 17, and a subtelomeric probe for 3p25. Sequencing of von Hippel-Lindau gene and analysis of the methylation status of the promoter region was also carried out in 2 tumors. Thirty-six tumors from 33 patients (mean age: 60.4 , range: 26 to 88; 17 men and 16 women) were studied. Three patients had bilateral tumors and 1 patient had von Hippel-Lindau disease. Follow-up was available in 60% (20/33) of the patients for a mean of 27.4 (range 1 to 85) months. No patient had evidence of the disease after surgery except for the patient with von Hippel-Lindau disease, who was alive with stable disease in the contralateral kidney. All 36 tumors were small (mean size 2.4 cm; range 0.9 to 4.5 cm) and low stage (pT1). The majority was cystic and had prominent fibrous capsule and stroma. The tumors were composed of variable amount of cysts, papillae, tubules, acini, and solid nests. The most characteristic histologic features were branching tubules and acini and anastomosing clear cell ribbons with low-grade nuclei. All tumors were strongly positive for CK7 and variably positive for CA9, but largely negative for CD10, and negative for α-methylacyl-CoA racemase and TFE-3. All but 1 tumor had no gains of chromosomes 7 and 17 and deletion of 3p. Only 1 tumor had low copy number gains of chromosomes 7 and 17. VHL gene mutation and promoter methylation were negative in 2 tumors analyzed. We show that these tumors, which we term as "clear cell tubulopapillary renal cell carcinoma," constitute a unique subtype in the spectrum of renal epithelial neoplasia based on their characteristic morphologic and immunohistochemical features.

A FISH study reveals ALK gains in colon cancer patients

The identification of oncogenic chromosomal alterations of anaplastic lymphoma kinase (ALK) in lung cancer and neuroblastoma has revealed ALK as a potential candidate for anticancer targeted therapy. Thus, ongoing clinical trials with ALK inhibitors are showing promising results. ALK chromosomal alterations in other solid tumors are controversial. The aim of this study was to analyze ALK translocations and gene copy number status in a series of colorectal cancer patients and correlate these findings with tumor pathological features including TNM staging, KRAS and BRAF mutations, as well as clinical outcome. ALK status was retrospectively evaluated by fluorescence in situ hybridization (FISH) with a break-apart ALK (2p23) probe (Abbott Molecular, Des Plaines, IL, USA) and centromeric probe of chromosome 2 (Abbott Molecular) in biopsies from colorectal cancer primary tumors, as were KRAS and BRAF by mutational analysis. Survival was analyzed by KaplaneMeier method. One hundred and four colorectal cancer patients were evaluated. ALK translocations were not observed; however, 39/104 (37%) of the patients had an increase in ALK gene copy number (3e5 copies), with the percentage of cells with ALK copy gain within a tumor ranging between 6% and 46% (mean 22%). There was no correlation between ALK gene copy number and KRAS mutational status, but BRAF mutations and ALK gain appear to be mutually exclusive, since the five patients with BRAF mutation had ALK disomic pattern. ALK gain was correlated with nodal status. Forty-three per cent of patients with positive nodal status had an increase in ALK copy number, while only 15% of patients with negative lymph nodes had ALK gain (p 5 0.05). The increase in ALK gene copy number was not correlated with overall survival. As a conclusion, the identification of ALK gene gain in colorectal cancer patients seems to be a relatively frequent genetic abnormality and is associated with tumors that have a more aggressive phenotype. Taking into account that ALK inhibitors are showing promising results in other types of tumors, it could be interesting to study ALK as a potential new therapeutic target for this tumor. Further evaluation of the biological meaning of ALK gain in colorectal cancer is ongoing. GERMLINE EPIMUTATION OF THE TUMOR SUPPRESSOR GENE PTPRJ IN EARLY ONSET FAMILIAL COLORECTAL CANCER

Application of touch FISH in the study of mosaic tetraploidy and maternal cell contamination in pregnancy losses

karyotype is a well established technique in the study of spontaneous miscarriages but is associated with selective overgrowth of maternal cells and other culture artefact (spp) such as tetraploidy, which could mask the true karyotype of the conceptus. 328 cases of pregnancy losses were studied by karyotype and Multiplex Ligation Dependent Probe Amplification technique. Touch FISH performed in non-cultured cells was used to evaluate the ploidy complement and sex discrepancies using centromeric probes for chromosomes X, Y and 18. touch FISH confirmed 13 cases of maternal contamination, identified a triploidy and a monosomy X. True tetraploidy was confirmed in 7/14 cases studied. touch FISH protocol is extremely accurate in the distinction of genuine mosaicism from tissue culture artifacts namely in cases with suspicion of tetraploidy and can be used to confirm maternal cell contamination in cases with sex discrepancy between karyotype and MLPA.

Renal oncocytoma: a comparative clinicopathologic study and fluorescent in-situ hybridization analysis of 73 cases with long-term follow-up

Clinical studies have confirmed that renal oncocytoma (RO) is a benign neoplasm with excellent prognosis. In diagnostically challenging cases of renal oncocytic epithelial neoplasms, fluorescent in-situ hybridization (FISH) is increasingly being used and its ability to distinguish RO from chromophobe renal cell carcinoma (ChRCC) has been documented. In this study, we evaluated the differential diagnostic contribution of FISH in cases of RO.Clinicopathologic data and glass slides from 73 patients with RO were reviewed; 20 cases of ChRCC were included for comparison. FISH analysis of formalin-fixed, paraffin-embedded sections was performed using centromeric probes for chromosomes 1, 2, 7 and 17. FISH analysis revealed ROs had frequent loss of signal for chromosome 1 (56%) and 17 (44%). Tumors with more than one loss were common (41%) and 10% cases showed loss of all chromosomes examined. A total of 18% cases did not show any abnormality.Our study shows that chromosomal abnormalities in both ROs and ChRCCs are common with frequent loss of chromosomes 1 and 17. No association was found between overall patient survival and the extent of chromosomal abnormalities. FISH results, even those showing significant chromosomal abnormalities, should not alter the primarily morphology-based diagnosis of RO.

Renal Cell Neoplasms of Oncocytosis Have Distinct Morphologic, Immunohistochemical, and Cytogenetic Profiles

This study was undertaken to elucidate the genetic patterns of the renal cell neoplasms of oncocytosis and to compare them with those found in cases with multiple oncocytomas. Three cases of renal oncocytosis and 6 cases of multiple oncocytomas were analyzed. Fluorescence in situ hybridization analysis was performed with centromeric probes for chromosomes 1, 2, 6, 10, and 17 that are typically lost in chromophobe renal cell carcinoma but not in oncocytoma. Immunohistochemistry for cytokeratin 7, parvalbumin, and S100A1 was performed in all cases. Eleven tumors were present in the 3 kidneys with oncocytosis. One of these was a classic chromophobe renal cell carcinoma. In the other 10, none showed any chromosomal losses, whereas 3 showed gains of all 5 chromosomes and 1 had gains of chromosomes 2 and 10. The chromophobe renal cell carcinoma showed losses of chromosome 1, 6, 10, and 17. Twelve of 14 tumors from the patients with multiple oncocytomas showed no loss of any of the chromosomes 1, 2, 6, 10, or 17 and 2 had loss of chromosome 1. All the tumors from kidneys with renal oncocytosis showed strong parvalbumin immunoreactivity, whereas cytokeratin 7 and S100A1 expression were variable. In summary, the renal cell neoplasms of oncocytosis seem to have distinct morphologic, immunohistochemical, and cytogenetic profiles and likely are a distinct entity, not closely related to oncocytoma or chromophobe renal cell carcinoma.