Document 15387
Issue 4 KREATECH NEWS IN THIS ISSUE… TWENTY-FOUR CHROMOSOME FISH IN HUMAN IVF EMBRYOS REVEALS PATTERNS OF POST-ZYGOTIC CHROMOSOME SEGREGATION AND NUCLEAR ORGANIZATION Dimitris Ioannou, Gothami Fonseka, Eric Meershoek, Alan Thornhill, Adulmawla Abogrein, Michael Ellis and Darren K. Griffin University of Kent, Kreatech, Digital Scientific and London Bridge Fertility, Gynaecology and Genetics Centre. In the May issue of Chromosome Research this year, Dimitris Ioannou and ....... Read more on page 5 P53 / MPO “ISO 17Q” FISH PROBE IN CHRONIC LYMPHOCYTIC LEUKEMIA ROUTINE DIAGNOSTICS DETECTION OF NUP98 GENE REARRANGEMENTS BY FLUORESCENT IN SITU HYBRIDIZATION Lana Harder, MD, PhD Institute of Tumour Genetics North, Kiel, Germany Susana Lisboa, Manuel R. Teixeira Department of Genetics, Portuguese Oncology Institute, Porto, Portugal REPEAT-FREE™ POSEIDON™ BCR/ABL1 t(9;22) PRODUCT RANGE Read more on page 8 Chronic lymphocytic leukemia (CLL) is a disease with a highly heterogeneous clinical course. Aberrations of the P53 pathway are increasingly recognized as one of the most important biological risk factors. Deletions of the short arm of chromosome 17 resulting in loss of one P53 allele occur in 8-10% of German CLL patients [1]. In other populations, the incidence can be higher, up to 16% [2]. Furthermore, P53 deletions were detected by FISH in 7% of multiple myeloma [3], in approximately 5% of myelodysplastic syndromes [4] and in up to 40% of complex aberrant acute myeloid leukemias [5]. In addition, P53 aberrations are recurrent abnormalities in almost all solid and hematologic cancers [6]. Read more on page 2 Nucleoporin 98 gene (NUP98) rearrangements have been identified in a wide range of hematologic malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia in blast crisis (CML-bc), myelodysplastic syndrome (MDS) and bilineage/ biphenotypic leukemia [1]. So far, NUP98 has been found to be rearranged with up to 28 different partner genes, resulting in in-frame fusion genes [1, 2]. The clinical course of patients with NUP98 gene rearrangements seems to be aggressive and presents a poor outcome [1], thus being important to recognize patients harboring such rearrangements. Read more on page 4 DEVELOPMENT AND VALIDATION OF A REPEAT-FREE™ DNA-FISH ASSAY TO DETECT FGFR1 GENE LOCUS AMPLIFICATION Read more on page 12 OTHER NEWS Please contact us or your local distributor for the previous issue of KREATECH NEWS You can also visit our website: www.kreatech.com www.kreatech.ccom IN THIS ISSUE SH IN BIOCHIPS FISH ITT TAKES TWO ISH4U FISH IN THIS ISSU E… DETECTION OF PROG NOSTICALLY RELEVANT GENETIC ABNORMAL CHILDHOOD ITIES IN B-CELL PRECU LYMPHOBLAS RSOR ACUTE TIC LEUKA EMIA By Prof. Christine J Harris Schwab, Dr. Amy Erhorn on, Ms. Claire Leukaemia . Research Cytogenetics Newcastle University, Group, Newcastle-up on-Tyne, UK The abnorm alities with the most impact on significant treatment and manag childhood ement of B-lineage acute lymph leukaemia oblastic are t(4;11) (q21;q23)/ t(9;22)(q34;q MLL-AFF1 11)/BCR-A , BL1 low hypodi ploidy for high and near-haploidy/ risk stratifi Read more cation…... on page 2 GATA 4 (8p23 ) MICRODELET IN CONG ION ENITAL HEART FLUORESCEN DIAPHRAGM DEFECT & CE IN SITU ATIC HERNI TO REFIN A E THE DIAGN HYBRIDISATION OSIS OF BONE SOFT TISSU By Prof. Damie E TUMOURS AND n Sanlaville, Laboratoire MD, PhD, de Cytogé nétique By Karoly Constitutionn Szuhai MD elle, CHU PhD 1, Hans and Pancra de Lyon, France J Tanke PhD 1 s C.W. Hogen . 1 Department doorn MD, Patients with 8p23 PhD 2. of Molecu 2 lar Cell Biolog microdeletion Department mild facial y and of exhibit dysmorphism Medical Center Pathology, Leiden Univer cephaly, enophtandem including duplication , Leiden, The sity 1549 by talmia , micrognathia microCHRONIC Netherlands. fusion of BRAF-KIAA rate intelle sho showing tissue LYMPHOCYTI , modectual deficie diatric glioma C LEUKEMIA FISH ANALY Primary hybridized to pediatric problems , Fusion Probe SIS (they display ourncy, U FISH4behav iour hetero bone and soft tissue 1549 (7q34) Triple-Color aboutsudden POSEIDON™ USING REPEAT-FREE chang tumours are geneous FISH4U BRAF-KIAA and es read more you can ™ DNA PROBE group of 7 of this newsletteraggres of behav iour,received manyextreme benign Read more tumours includi a S. (2R2G1F3B). utedd On page program we have with outbur on page 8 contribute ng in this sts of mesen and malignant lesions the launch of this siveness and Si Since destruand that has been kindly d probes and . They have ctive behav chymal origin, ico- program. customize with an article ent ofmalfor e de Physicoand iour) mations. a someti This issue will start fo the developmare for ANNOUNCIN to origina Viovy from Laboratoir Two works how this program the requests malformation freque aboutntly te from mesen mes hypothesised Prof. dr. Jean-Louis G THE THERM associated This article describes you can find more s [1]. The by the group of artic article France. ELITE chyma in short OBRITE ® deletio with Paris ; AUTOMATION l stem cells case Curie, diagnosis 8p23 micron, namel chips. To showcase of these y congenital FOR FISH Chimie Curie, Institut ccan do for you. (CHD) Read more challenging using monolithic TESTING. lithic what it heart defect and congen on page 10 in daily clinicop lesions may be of FISH protocols using monolithic protocols ital miniaturization FISH as diaphr atholog of they are relative (CDH) [1]. agmatic hernia ical practic re for of miniaturization The first a procedure e, ly rare: sarcom the possibilities have developed mesenchymal Happy reading! with an 8p23s team description of as, malign of professor Viovy Diagnostic deletio a person tumours, are ant cell line as a model. KREATECH chips the group The SK-BR-3 n Please the cancer was in 1988. less than 2% Sincerely, more than s, 100 distinc contact us of HER2 using openss new Sincere 50 cases of of all To date or your local t subtypes the molecular typing ic FISH platform interstitial Furthermore 8p23.1 deletio distributor are defined for the previou g cells. or terminal that such a microfluid morphologic ns have been s issues of The results show screening on circulating criteria of malign [2] used for deletions KREATECH reported. early-stage cancer You can also occur in equal NEWS ancy 8p23 applica other cancer types possibilities for routine visit our websit frequency and female are not always ble [2] For ULS™ e: in males s and across www.kreatech the use of the instance, “ominous” all ethnic DIAGNOS exact TICS read more on .com. .c com. com histologically frequency ion by KREATECHOGY DNA PROBES looking article you can of this microd groups. The (i.e. Following this HEMATOL known ive genomic hybridizat y for nodular fasciiti lesions may be benign KREA KREATECHeletion ogy although y for comparat technolog is not NEWS TECH s or ancien KREATECH labeling technolog REPEAT-FREE™ and decept been report it varies betwe is hasNEWS t schwannoma) NEWS ively “bland application of the ed that en 1/55.0 ” lesions arrayGCH and the malignant 00 and 1/7000 births [2]]... may have ry FISH. behavi Read confirmato our t a live sarcom (i.e. low grade more on page subsequen a or epithel fibromyxoid 4 ioid sarcom Read more a) [2].… on page 5 IIOOONN LATEST EDITION WELCOME TO THE NEWSLETTER! R!! OF THE KREATECH OTHER NEW S Issue 1 INN THIS ISSUE WELCOM OF THE E TO THE KREATEC LATEST H NEWSLETEDITION TER! BRAF-KIAA1549 (2R2G1F3B). All prostate cancers cance become essentially within a few years(7q34) resistant to endocrine therapy. Triple-Color, ther This stage ofThis disease issue is known as Fusion Resistant Prostate Prostat Cancer (CRPC).byRemarkablywill start CastrationProbe the group in approx. 30% of hybridized the with CRPC cases the most m important mechanism Chimie seems an article of to pediatric Androgen Curie, Prof. dr. Receptor (AR) overexpression overexpre miniaturization caused by high Institut Jean-Louis that has glioma level amplification ofCurie, the AR gene.Viovy been tissue Read more on page 5 on The the kindly showing Role ofpossibilities from Paris the AndrogenofReceptor FISH in Prostate fusion Cancer by Prof. protocols in France. Laboratoire contributed Pro Trapman, Erasmus chipsMedical of BRAF-KIAA1549 of miniaturization Centre, the group This Rotterdam. using de PhysicoOn page the molecular monolithic article describes of professor by tandem of FISH program. 7 of this Translocati Translocations TheMLL involving the typing newsletter the results Viovy protocolschips. To showcase duplication gene and its numerous requests Since the have show oneofofHER2 partner genes you can ge belong to possibilities developed using monolithic the recurring cytogenetic using that for the launch of read short the SK-BR-3 for routine such aberrations in Acute Myeloid Leukemia. developmentthis program more article a procedure a microfluidic MLL translocations about exert prognostic you can what early-stage cell line influence on response Following our FISH we have it can of customized for to induction therapy and FISH as a cancer do for find more 4U received survival. this The screeningplatform model. effect depends d about you. stronglylabeling on the partner article probes many opens gene. Therefore how Happy you can clarifying arrayGCH technology on circulating new this program and in exactly the aberration partner reading! read is of this Sincerely, for forcomparative further more therapy subsequentandimportance cells. works decisio Continued on decisions. the application on the page 7 The KREATECH and confirmatory genomic use of the of the Diagnostics ULS™ REPEAT-FREE™hybridization FISH. We are a very grateful to these authors for their contribution team to this new newsletter technology by and we hope that you will enjoy it. for Please KREATECH contact Yo sincerely, Kreatech Diagnostics Yours HEMATOLOGYDIAGNOSTICS Please contact our Pathology us or You Brochureyour local can also distributor visit and our website:Catalogue for 2011-2012. www.kreatech.com sincerely, KREATECH CATALOGUE DIAGNOSTICS 2011 - 2012 te: www.kreatech.com rvice@kreatech. com KREATECH SOLID DIAGNOSTICS TUMORS omerservice@kreatech.com w.kreatech.com KREATECH rvice@kreatech.com ech.com Published Januari 2011 Reader, CANCER PROSTATE IN AML CANCER Welcome issue to our FISH we present latest edition DNA provides probes our latest of the you and releases Kreatech with our FISH4U of REPEAT-FREE the Newsletter. We probe are opinion proud of choice.Custom In this Probe POSEIDON leaders to present Service in Europe. a selection Promising which To give of Crizotinib results you informative have (NSCLC) (PF-02341066) a preview been articles In Situ patients obtained on these from Hybridization carrying for treatment with the articles: diagnosis ALK the Pathology of inhibitors of ALK is most fusion gene non-small Department, such likely rearrangement cell as the ALK-EML4. lung All prostate best cancer Hôpital adapted “Fluorescent to endocrine cancers Cochin, in NSCLC” method Resistant Paris. cited therapy. become Read by Dr. for Prostate essentially CRPC This more cases stage on pageJust (AR) within the mostCancer overexpression (CRPC). of disease 2 a few Read important Remarkablyis known years more Cancer caused resistant on mechanism in approx.as Castrationby Prof. page 5 on by high Trapman, The Role level seems Androgen30% of Translocations amplification Erasmus of the Androgen the partner Receptor of Medical involving Receptorthe AR in Acute genes Centre, gene. in Prostate influence Myeloidbelong the MLL Rotterdam. to gene Leukemia.the recurring effect on and response one exactly depends MLL of its cytogenetic translocations strongly to induction numerous decisions.the aberration on Continued partnerthe partner therapy exert aberrations prognostic gene. and survival. on page is of importance Therefore 7 The for further clarifying and to we hope these therapy authors that for their you will Kreatech enjoy contribution Diagnostics it. to this We are very newsletter grateful Yours E-mail: customerservice@kreatech.com Website: www.kreatech.com ail: customerse DIAGNOSTICS CATALOGUE 2011 © 2011 KREATECH Diagnostics FISH DIAGNOSTICS Januari DIAGNOSTICS DIAGNO OSTICS C hed RF™ POSEIDON™ SOLUTIONS FOR HEMATOLOGY TUMOR FI FISH 2011 - 2012 RF™ P SOL SOLUTIONS O UT POSEIDON™ FOR SOLID TUMOR FISH DIAGNOSTICS 1 Published Januari 2011 IN THIS ISSUE ALK REARRANGEMENT ANDROGEN MLL RECEPTOR IN LUNG TRANSLOCATIONS IN Dear CATALOGUE CAT ATALOGUE 2011 - 2012 ATALOGUE 2011 - 2012 CAT CATALOGUE © 2011 KREATECH Diagnostics KREATECH NEWS . RF™ ™ POSEIDON™ SOL SOLUTIONS UTIONS NS FOR SOLID I Issue Please contact us or your local distributor for our Pathology Brochure and Catalogue 2011-2012. 12. You can also visit our website: www.kreatech.com m KREATECH CH DIAGNOSTICS KREATECH DIAGNOSTICS RF™ ™ POSEIDON™ SOLUTIONS SOLUUTIONS NS FOR SOLID I TUMOR FISH I DIAGNO DIAGNOSTICS OSTICS C DNA PROBES Publis KREATECH DIAGNOSTICS SOLID TUMORS of KREATECH us or your local NEWS You can also visit and for the distributor KREATECH DIAGNOSTICS for KREATECH CATALOGUE 2011 - 2012 ourDIAGNOSTICS SOLID TUMORS website: HEMATOLOGY the previous Brochure. www.kreatech.com issue ics Yours sincerely, Kreatech Diagnostics Diagnost We are very grateful to these authors for their contribution to this newsletter and we hope that you will enjoy it. KREATECH Translocations involving the MLL gene and one of its numerous partner genes belong to the recurring cytogenetic aberrations in Acute Myeloid Leukemia. MLL translocations exert prognostic influence on response to induction therapy and survival. The effect depends strongly on the partner gene. Therefore clarifying exactly the aberration partner is of importance for further therapy decisions. Continued on page 7 KREATECH DIAGNOSTICS CATALOGUE 2011 - 2012 2 Promising results have been b obtained with ALK inhibitors such as Crizotinib (PF-02341066) (PF-0234106 for treatment of non-small cell lung cancer (NSCLC) patients carrying carry the fusion gene ALK-EML4. “Fluorescent In Situ Hybridization is most likely the best adapted method for the diagnosis of ALK rearrangement in NSCLC” cited by Dr. Just Pathology Department, Departme Hôpital Cochin, Paris. Read more on page 2 FISH4U All prostate cancers become essentially within a few years resistant to endocrine therapy. This stage of disease is known as CastrationResistant Prostate Cancer (CRPC). Remarkably in approx. 30% of the CRPC cases the most important mechanism seems Androgen Receptor (AR) overexpression caused by high level amplification of the AR gene. Read more on page 5 on The Role of the Androgen Receptor in Prostate Cancer by Prof. Trapman, Erasmus Medical Centre, Rotterdam. Pleasee contact us or your local distributor for 2011-2012. our Patholog Pathology Brochure and Catalogue gue 2011 -2012 -2012. You can also visit our website: www.kreatech w.kreatech.com .co www.kreatech.com IN THIS ISSUE FISH IN BIOCHIPS IT TAKES TWO FISH4 U We are proud to present a selection of informative articles from opinion leaders in Europe. Europe To give you a preview on these articles: Promising results have been obtained with ALK inhibitors such as Crizotinib (PF-02341066) for treatment of non-small cell lung cancer (NSCLC) patients carrying the fusion gene ALK-EML4. “Fluorescent In Situ Hybridization is most likely the best adapted method for the diagnosis of ALK rearrangement in NSCLC” cited by Dr. Just Pathology Department, Hôpital Cochin, Paris. Read more on page 2 Issue ALK REARRANGEMENT NTT INN LLUNG CANCER ANDROGEN RECEPTOR ORR INN PROSTATE CANCER MLL TRANSLOCATIO TRANSLOCATIONS ONNS NS IN AML Dear Reader, Welcome to our latest edition of the Kreatech Newsletter. In this issue we present our latest rreleases of REPEAT-FREE POSEIDON FISH DNA probes and our F FISH4U Custom Probe Service which provides you with the probe of choice. We are proud to present a selection of informative articles from opinion leaders in Europe. To give you a preview on these articles: FISH DIAGNOSTICS RF™ POSEIDON™ FOR HEMATOLOGY SOLUTIONS Issue 1 IN THIS ISSUE ALK REARRANGEMENT IN LUNG CANCER ANDROGEN RECEPTOR IN PROSTATE CANCER MLL TRANSLOCATIONS IN AML Dear Reader, Welcome to our latest edition of the Kreatech Newsletter. In this issue we present our latest releases of REPEAT-FREE POSEIDON FISH DNA probes and our FISH4U Custom Probe Service which provides you with the probe of choice. i prevvioouss issue r for the previous or your local distributo hure.. Please contact us OGY Brochure and for the HEMATOL of KREATECH NEWS tech.com. our website: www.krea You can also visit Issue 3 KREATECH NEWS Issue 2 KREATECH NEWS © 2011 FGFR1 (8p11) / SE 8 probe hybridized to SCC NSCLC and BC tissue KREATECH NEWS P53 / MPO “ISO 17Q” FISH PROBE IN CHRONIC LYMPHOCYTIC LEUKEMIA ROUTINE DIAGNOSTICS Lana Harder, MD, PhD Institute of Tumour Genetics North, Kiel, Germany CLINICAL BACKGROUND OF CLL WITH P53 DELETIONS Chronic lymphocytic leukemia (CLL) is a disease with a highly heterogeneous clinical course. Aberrations of the P53 pathway are increasingly recognized as one of the most important biological risk factors. Deletions of the short arm of chromosome 17 resulting in loss of one P53 allele occur in 8-10% of German CLL patients [1]. In other populations, the incidence can be higher, up to 16% [2]. Furthermore, P53 deletions were detected by FISH in 7% of multiple myeloma [3], in approximately 5% of myelodysplastic syndromes [4] and in up to 40% of complex aberrant acute myeloid leukemias [5]. In addition, P53 aberrations are recurrent abnormalities in almost all solid and hematologic cancers [6]. Lana Harder, MD, PhD in her new practice (founded January 1st, 2012 at the Institute of Tumour Genetics North). It is well established that deletions of P53 are known to be associated with poor response to therapy (with purine analog refractory), aggressive disease and shorter survival [1, 7, 8, 9]. Therefore, estimation of genetic risk parameters has become increasingly important [10]. In CLL patients with loss/mutation of P53, so-called “high risk CLL” an allogeneic stem cell transplantation (SCT) should be considered [9]. Nowadays, P53 status in CLL patients should be identified prior to treatment [9, 11]. Cytogenetic analyses and fluorescence in situ hybridization (FISH) are helpful tools for investigation of the P53 status at initial diagnosis, at follow up, and especially during disease progression [12]. P53 can be lost due to pure intrachromosomal deletions in 17p, monosomy 17, unbalanced translocations involving the short arm of chromosome 17 and formation of an isochromosome 17q. Unbalanced translocations involving 17p and isochromosomes 17q with breakpoints between 17p10 and 17p11.2 are recurrent events triggered by low-copy DNA repeats located in 17p10 to 17p12 [13]. 2 DESIGN OF P53 FISH PROBES The commonly used FISH probe for detecting the P53 gene locus is the P53 probe combined with the centromeric region of chromosome 17. We prefer the use of the FISH probe P53 in combination with the MPO probe at 17q22. The P53 / MPO probe combination has the advantage that the MPO gene locus probe displays more or less similar signal intensity as the specific P53 probe, whereas any commercially available FISH probe for P53 and the centromeric region of chromosome 17 can give a stronger signal of the centromeric region due to the repetitive character of this region. In our hands the P53 / MPO probe is easier to use, especially in tumor samples with poor cell morphology as one can better distinguish a true P53 deletion from a reduced signal of P53 which possibly could lower the amount of false positive results. A second advantage of the P53 / MPO probe combination is the possibility to detect isochromosomes 17 very easily by FISH: An isochromosome 17q results in a deletion of the short arm of chromosome 17 resulting in a loss of one P53 gene signal and a gain of the long arm of chromosome 17 leading to an additional MPO gene signal. Using the two diagnostic criteria, loss of one P53 signal and gain of one MPO signal, gives a high sensitivity for detection of a tumor clone with an isochromosome 17q. Issue 4 p53 (17p13) / MPO (17q22) “ISO 17q” probe hybridized to peripheral blood of a CLL patient with a 17p- deletion (1 green and 2 red signals). FISH PROBE EVALUATION Every lab should establish their own cut off levels for the detection of a P53 deletion as well as for the gain of the MPO locus in interphase cells. In our lab, blood samples from five healthy persons served as negative controls. For each blood sample 200 interphase nuclei were evaluated for the determination of the diagnostic thresholds of the P53 and MPO probes. Cut off levels for a loss or gain of the P53 and MPO gene locus were calculated as mean of false positive nuclei plus three standard deviations, respectively. REFERENCES [1] Döhner H. et al. 2000, Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med 343:1910-1916. [2] Xu W. et al. 2008, Prognostic significance of ATM and TP53 deletions in Chinese patients with chronic lymphocytic leukemia. Leuk Res 32:1071-1077. [3] Walker BA. et al. 2010, A compendium of myeloma-associated chromosomal copy number abnormalities and their prognostic value. Blood 116:56-65. [4] Haase D. et al. 2007, New insights into the prognostic impact of the karyotype in MDS and correlation with subtypes: evidence from a core dataset of 2124 patients. Blood 110:4385-95. [5] Rücker FG. et al. 2012, TP53 alterations in acute myeloid leukemia with complex karyotype correlate with specific copy number alterations, monosomal karyotype, and dismal outcome. Blood 119:211421-21. [6] Swerdlow SH. et al. 2008, WHO classification of Tumors of hematopoietic and lymphiod tussue. IARC: Lyon. [7] Cordone I. et al. 1998, p53 expression in B-cell chronic lymphocytic leukemia : a marker of disease progression and poor prognosis. Blood 91:4342-4249. [8] Haferlach C. et al. 2010, Toward a comprehensive prognostic scoring system in chronic lymphocytic leukemia based on a combination of genetic parameters. Genes Chromosomes Cancer 49:851-9. [9] Zenz T. et al. 2012, Risk categories and refractory CLL in the era of chemoimmunotherapy. Blood 119:4101-4107. p53 (17p13) / MPO (17q22) “ISO 17q” probe hybridized to peripheral blood of a CLL patient with an isochromosome 17 (1 green and 3 red signals). [10] Gonzalez D. et al. 2011, Mutational status of the TP53 gene as a predictor of response and survival in patients with chronic lymphocytic leukemia: results from the LRF CLL4 trial. J Clin Oncol 29:2223-9. [11] Pettitt AR. et al. 2012, Alemtuzumab in combination with methylprednisolone is a highly effective induction regimen for patients with chronic lymphocytic leukemia and deletion of TP53: final results of the national cancer research institute CLL206 trial. J Clin Oncol 30:1647-55. [12] Delgado J. et al. 2012, Chronic lymphocytic leukaemia with 17p deletion: a retrospective analysis of prognostic factors and therapy results. Br J Haematol 157:67-74. [13] Fink SR. et al. 2006, Loss of TP53 is due to rearrangements involving chromosome region 17p10 approximately p12 in chronic lymphocytic leukemia. Cancer Genet Cytogenet. 167:177-81). To try out our P53 probes please contact your local representative 17p13 D17S2151 D17S960 17q22 330 KB P53 17 D17S634 MPO 400 KB SHGC-144222 17 Ordering information Tests Cat# ON p53 (17p13) / MPO (17q22) “ISO 17q” 10 KBI-10011 ON p53 (17p13) / SE 17 10 KBI-10112 ON p53 (17p13) / SE 17 20 KBI-12112 ON p53 (17p13) / ATM (11q22) 10 KBI-10114 ON p53 (17p13) / SE 17 (tissue) 10 KBI-10738 3 KREATECH NEWS DETECTION OF NUP98 GENE REARRANGEMENTS BY FLUORESCENT IN SITU HYBRIDIZATION Susana Lisboa, Manuel R. Teixeira Department of Genetics, Portuguese Oncology Institute, Porto, Portugal 11p15 SHGC-84145 530 KB RH75370 GAP 455 KB Nucleoporin 98 gene (NUP98) rearrangements have been identified in a wide range of hematologic malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia in blast crisis (CML-bc), myelodysplastic syndrome (MDS) and bilineage/ biphenotypic leukemia [1]. So far, NUP98 has been found to be rearranged with up to 28 different partner genes, resulting in in-frame fusion genes [1, 2]. The clinical course of patients with NUP98 gene rearrangements seems to be aggressive and presents a poor outcome [1], thus being important to recognize patients harboring such rearrangements. Fluorescent in situ hybridization (FISH) using a dual color, break apart probe flanking the NUP98 gene is a rapid method that allows detection of rearrangements involving this gene, regardless of the fusion partner. We have used the REPEAT-FREE™ (RF) POSEIDON™ NUP98 (11p15) Break Probe (cat# KBI-10311) to test for the presence of NUP98 gene rearrangements in two AML cases with 11p15 abnormalities as detected by karyotype analysis and one AML case, previously published by our group [3], harboring a NUP98 gene fusion. As a normal control, we used three samples obtained from peripheral blood cell culture of normal donors. Slides were prepared fresh from cultured cells fixed with methanol:acetic acid and were pretreated with 2 x SSC/ 0.5% Igepal at 37 °C for 20 minutes (min). Co-denaturation was performed at 80 °C for 8 min followed by hybridization at 37 °C in humidified chamber for 16 hours. Post- hybridization washes were done using 0.4 x SSC/ 0.3% Igepal at 74 °C for 2 min and 2 x SSC/ 0.1% Igepal for one min, at room temperature. DAPI was applied as a counterstain and results were evaluated with a Zeiss Axioplan 2 fluorescence microscope. For each sample, 100 intact non-overlapping nuclei were scored. We have detected a normal signal pattern (two fusion signals) in all the NUP98 D11S4525 410 KB SHGC-79113 11 Ordering information Tests Cat# ON NUP98 (11p15) Break* 10 KBI-10311 * Available soon control samples. The two cases with 11p15 karyotype abnormalities and the case with a known NUP98 gene rearrangement presented abnormal signal patterns, shown by the presence of a fusion signal and isolated green and red signals. The use of the break apart NUP98 FISH probe allows the screening of patients for rearrangements involving this gene, therefore making it possible to better characterize and monitor such patients. REFERENCES [1] Gough SM. et al. 2011, NUP98 gene fusions and hematopoietic malignancies: common themes and new biologic insights. Blood 118: 6247-6257. [2] Nebral K. et al. 2005, Screening for NUP98 rearrangements in hematopoietic malignancies by fluorescence in situ hybridization. Haematologica 90: 746-752. [3] Cerveira N, Correia C, Dória S, Bizarro S, Rocha P, Gomes P, Torres L, Norton L, Borges BS, Castedo S, Teixeira M. 2003, Frequency of NUP98-NSD1 fusion transcript in childhood acute myeloid leukaemia. Leukemia 17: 2244-7. RF POSEIDON NUP98 (11p15) Break Probe hybridized to AML patient sample showing a rearrangement of 11p15 involving the NUP98 gene (1 Fusion, 1 Red, 1 Green signal). 4 Issue 4 TWENTY-FOUR CHROMOSOME FISH IN HUMAN IVF EMBRYOS REVEALS PATTERNS OF POST-ZYGOTIC CHROMOSOME SEGREGATION AND NUCLEAR ORGANIZATION Dimitris Ioannou, Gothami Fonseka, Eric Meershoek, Alan Thornhill, Adulmawla Abogrein, Michael Ellis and Darren K. Griffin University of Kent, Kreatech, Digital Scientific and London Bridge Fertility, Gynaecology and Genetics Centre. INTRODUCTION In the May issue of Chromosome Research this year, Dimitris Ioannou and colleagues from Darren Griffin’s lab at the University of Kent published a manuscript describing the use of a 24 chromosome screen, previously described in Ioannou et al (2011), which makes use of Kreatech fluorescence in situ hybridization (FISH) probes, on human in vitro fertilization (IVF) embryos (Ioannou et al 2012). Darren Griffin was in fact involved in the first introduction of FISH as a tool in the IVF world in the early 1990s as a means of determining sex for preimplantation genetic diagnosis (PGD) to treat couples at risk of transmitting sex-linked disorders such as Duchenne Muscular Dystrophy. FISH was later used for the diagnosis of unbalanced chromosome translocations and for screening for aneuploidy. The latter became the infamous preimplantation genetic screening (PGS) that courted much controversy in both the scientific and popular press. This is because early indications that PGS would be effective were challenged by controlled and randomized trails that suggested that PGS actually made IVF success rates worse. In part due to this bad press, FISH-based technologies were replaced by microarraybased technologies for PGS. FISH analysis can still be valuable, however in analyzing embryos not used to establish a pregnancy, as it gives valuable information on the accuracy of the original PGS result and an insight into very early human development. Although it is theoretically possible to use microarray technologies cell-by-cell on early IVF embryos, such an approach would prove to be very costly. Thus any “followup” analysis by microarrays would be done on the whole embryo thereby neglecting individual cell analysis and limiting any analysis of chromosome mosaicism. FISH is a very good tool for investigating mechanisms of both mitotic chromosome segregation errors and chromosome position. In this regard the message is “the more chromosomes analysed the better” with all 24 chromosomes being optimal. TABLE 1 THE PROBES USED AND THEIR LOCI IN EACH OF THE MULTI-COLOUR PROBE MIXES FLUOROCHROME LAYER A: CENTROMERICPROBES LAYER B: LAYER C: LAYER D: UNIQUE CENTROMERIC PROBES CENTROMERIC PROBES SEQUENCE PROBES PlatinumBright™ 405 Dark blue SE7 (7p11-q11) SE11 (11p11-q11) SE18 (18p11-q11) CD37 (19q13) PlatinumBright ™ 415 Light blue(aqua) SE1 (1q12) SE9 (9q12) SE16 (16p11-q11) PDGFRB (5q33) PlatinumBright ™ 495 Green SE6 (6p11-q11) SE20 (20p11-q11) SE2 (2p11-q11) DSCR (21q22) PlatinumBright ™ 547 Light red/orange SE8 (8p11-q11) SE12 (12p11-q11) SEX (Xp11-q11) BCR (22q11) PlatinumBright ™ 590 Dark red SE3 (3p11-q11) SE10 (10p11-q11) SEY (Yp11-q11) RB (13q14) PlatinumBright ™ 647 Far red SE4 (4p11-q11) SE17 (17p11-q11) SE15 (15p11-q11) IGH (14q32) The fluorescent dyes with which they were labelled and the order in which they were hybridised is given (A, B, C, then D). Probes for chromosomes 1 and 9 were for highly repetitive heterochromatic regions below the centromere. SE satellite enumeration —the Kreatech trade name 5 KREATECH NEWS As mentioned, the authors (Ioannou et al. 2011) recently described the development of a 24 chromosome assay involving a series of six Kreatech fluorochrome-labeled probes and four rounds of hybridization. As proof of principle, they demonstrated that it could be used on lymphocytes, sperm and IVF embryo nuclei. Here, they demonstrate further that this approach has a dual applicability for the determination of aneuploidy and nuclear position of mostly centromeric loci on every chromosome in human IVF embryos at around days 5–6 of development. The main aims of the study were firstly to confirm or refute former studies that indicated mitotic non-disjunction was not the mechanism leading to post-zygotic aneuploidy, rather is was independent chromosome loss and gain. Second to ask which chromosomes are more likely to undergo gain or loss in preimplantation embryos, and finally to assay the nuclear positions of the loci recognized by the Kreatech probes. MATERIALS AND METHODS Material used in this study was mostly “follow up” aneuploid PGS cases, the collaborating clinics were the London Bridge Fertility Centre and the Lister Fertility Clinic. The Ioannou et al (2011) paper described a protocol that involved six Kreatech fluorochromes, namely PlatinumBright™405 (dark blue), 415 (light blue/aqua), 495 (green), 547 (light red/orange), 590 (dark red), 647 (far red) plus the DAPI counterstain in a four-stage probing and re-probing strategy. All probes for this protocol were synthesized by Kreatech Diagnostics using the Universal Linkage System (ULS™) for labeling, including six unique sequence targets for chromosomes 5, 13, 14, 19, 21 and 22 and the remaining 18 centromeric probes (See table 1). Human IVF embryo nuclei were fixed to slides by standard protocols. Then slides were washed in PBS for 2 minutes (min) and dehydrated and dried using an ethanol series. Pepsin treatment removed excess protein (1 mg/ml pepsin in 0.01 M HCl, 20 min at 37 °C), then the slides were rinsed in distilled water and PBS, followed by a paraformaldehyde (1% in PBS) fix at 4 °C for 10 min, then another PBS and distilled water wash and an ethanol dehydration and dry. The four probe combinations described by Ioannou et al. 2011 were dissolved in hybridization mix of Kreatech. It was important to predenature the probes at 73 °C for 10 min before application on the slide. Then co-denaturation of probe and chromosomes at 75 °C for 90 seconds (s) in a “Thermobrite-StatSpin” went ahead of hybridization at 37 °C. The hybridization period for the first three rounds of hybridization (centromeric probes) was for 30 min, whereas for the final round, it was overnight. Post-hybridization washes were for 1 min 30 s in 0.7× SSC, 0.3%Tween 20 at 72 °C followed by a 2 min in 2×SSC at room temperature. Slides were mounted in Vectashield containing 0.1 ng/μl of DAPI (Vector labs) before microscopy and image analysis. After analysis and image capture, slides were washed in 2×SSC at room temperature to remove the coverslip and then washed for 30 s in distilled water (72 °C) to remove the bound probe. An ethanol series preceded air-drying before continuation to the next round of hybridization. The protocol was the same for the second, third and final rounds with the following exceptions: The overnight hybridization time for the final round (previously mentioned), pepsin and paraformaldehyde treatment were only required for the first round; the post-hybridization wash time was reduced with every round from 90 s (first round of hybridization) to 50–60 s (second round) to 30 s (third and final rounds). Microscopy analysis was performed on an Olympus BX-61 epifluorescence microscope equipped with a cooled CCD camera (by Digital Scientific—Hamamatsu Orca-ER C4742-80) and using the appropriate filters. To enable analysis of the fluorochromes for image acquisition two communicating filter wheels (Digital Scientific UK) with the appropriate filters were used. The recommended filters by the probe manufacturer can be found here: http://www.kreatech.com/rest/customer-service-support/ technical-support/fluorophores-and-filter-recommendation.html and the image capture system was SmartCapture (Digital Scientific UK). Fig.1Blastomere nucleus after four rounds of hybridisation, scales bars=10μm. a DAPI only, showing retention of nuclear structure b Same nucleus with final probe set signals shown— chromosome 19 in blue, chromosome 5 in aqua,chromosome 21 in green, chromosome 22 in yellow, chromosome 13 in red, chromosome 14—far red fluorochrome pseudocoloured purple. c Same nucleus with probes from the other three rounds super imposed in Adobe Photoshop—note position and copy number of chromosomes 5, 13, 14, 19, 21and 22 can still be observed. 6 Issue 4 RESULTS AND DISCUSSION Analysis of 17 embryos by this newly developed approach gave strong signals for all chromosomes; it revealed chromosome copy number for each human chromosome per nucleus for each embryo and the nuclear positions of all the loci that were probed. As all embryos were ‘follow-up” (and most had a prior abnormal PGS result) the expected high levels of chromosome abnormalities were seen and no single nucleus displayed a normal chromosome complement. Moreover all showed evidence of mosaicism. There were certain patterns that emerged however. For instance chromosome loss appeared more common than both chromosome gain and apparent mitotic nondisjunction (thus confirmation of the first initial hypothesis). Next, in terms of chromosome position, the centromeric probes tended preferentially to occupy the nuclear centre. Where we had a prior day 3 biopsy PGS result, it was confirmed, at least partly, by 24 colour FISH in the majority of instances. In conclusion, therefore, the authors presented a new approach for assessing aneuploidy and chromosome position in human IVF embryo nuclei that retains the advantages of FISH while circumventing its former limitations (i.e. it could previously only assay a small number of chromosomes). The great value is its application for the providing insight into the cytogenetics of early human development. Some of the advantages over microarray array-based approaches lie in the fact that it is, in comparison to microarrays, inexpensive, and it gives a cell by cell analysis, which, while possible by microarrays, is practically and financially not feasible given the numbers of nuclei that need to be analysed. The added benefit is that the approach provides extra insight into the role of chromosome position (nuclear organization) in early human development. REFERENCES [1] Ioannou D, Fonseka KGL, Meershoek EJ, Thornhill AR, Abogrein A, Ellis M, Griffin DK, 2012, Twenty-four chromosome FISH in human IVF embryos reveals patterns of postzygotic chromosome segregation and nuclear organisation, Chromosome Research 20: 447-60. [2] Ioannou D, Meershoek EJ, Thornhill AR, Ellis M, Griffin DK, 2011, Multicolour interphase cytogenetics: 24 chromosome probes, 6 colours, 4 layers. Molecular and Cellular Probes 25:199– 205. PRODUCT AND ORDERING INFORMATION Product Description Tests Cat# PreimpScreen PolB (13,16,18,21,22) Five-color FISH-mix consisting of DNA probes specific for chromosomes 13, 16, 18, 21, and 22 20 KBI-40050 PreimpScreen Blas (13,18,21,X,Y) Five-color FISH-mix consisting of DNA probes specific for chromosomes 13, 18, 21, X, and Y 20 KBI-40051 MultiStar 24 FISH FISH probe panel for visualizing all 24 chromosomes (including the four panels KBI-40061, KBI-40062, KBI-40063, and KBI-40064) 10 KBI-40060 MultiStar FISH Panel 1 FISH panel of centromeric probes for chromosomes 1, 3, 4, 6, 7, and 8 10 KBI-40061 MultiStar FISH Panel 2 FISH panel of centromeric probes for chromosomes 9, 10, 11, 12, 17, and 20 10 KBI-40062 MultiStar FISH Panel 3 FISH panel of centromeric probes for chromosomes 2, 15, 16, 18, X, and Y 10 KBI-40063 MultiStar FISH Panel 4 FISH panel of unique sequence probes for chromosomes 5, 13, 14, 19, 21, and 22 10 KBI-40064 7 KREATECH NEWS REPEAT-FREE™ POSEIDON™ BCR/ABL1 t(9;22) PRODUCT RANGE The Philadelphia chromosome (Ph) is an abnormal chromosome 22 (der22) involved in a translocation with chromosome 9. The presence of the Ph chromosome is characteristic of Chronic Myelogenous Leukemia (CML), found in 95% of the cases. However, the presence of this characteristic t(9;22) BCR-ABL1 reciprocal chromosomal translocation is not solely specific for CML as it is also shown in about 25-30% of adult Acute Lymphoblastic Leukemia (ALL) cases and 2-10% of childhood ALL and occasionally in Acute Myelogenous Leukemia (AML). In the formation of the Ph translocation, the c-abl oncogene 1 (ABL1) on chromosome 9 and the breakpoint cluster region (BCR) on chromosome 22 fuse, generating two fusion genes: BCR-ABL1 on the Ph chromosome 22 and ABL1-BCR on the chromosome 9 (figure 1). The chimeric BCR/ABL1 fusion gene encodes a deregulated constitutively activated protein tyrosine kinase with profound effects on cell cycle, adhesion, and apoptosis. Understanding this process has led to the development of the drug imatinib mesylate (Gleevec™), a tyrosine kinase inhibitor. BCR-ABL1 BCR 22q11 der(22) 22 9 ABL 9q34 ABL1-BCR A In BCR there are three breakpoint regions, the major breakpoint cluster region (M-BCR), between exons 12 and 16 leading to the creation of the oncogene p210 BCR-ABL1 and the minor breakpoint cluster region (m-BCR), which maps the first intron of BCR and leads to the creation of the oncogene p185 BCR-ABL1. In CML, the breakpoint in BCR is mostly in the M-BCR located. Breaks in the m-BCR are most frequently associated with Ph-positive ALL. The micro BCR is very rare and only described in a few cases Fluorescence in situ hybridization (FISH) is used to confirm the presence of a BCR-ABL1 gene in the initial diagnosis of CML or Ph-positive ALL or AML. FISH has the advantage that it may detect cryptic BCR-ABL1 rearrangements not picked up by karyotyping and also possible deletions in the derivative 9 (der (9)) chromosome. FISH is also a valuable tool in determining the percentage patient's blood or bone marrow cells harboring the Ph chromosome and to monitor response to treatment and disease recurrence. der(9) Figure1: BCR/ABL1 t(9;22) Dual-Fusion Assay The REPEAT-FREE POSEIDON portfolio contains five different designs of the BCR/ABL1 t(9;22) probe each providing different details. BCR/ABL t(9;22) Dual-Color, Dual-Fusion BCR/ABL t(9;22) Triple-Color, Dual-Fusion BCR/ABL t(9;22) Dual-Color, Single-Fusion 8 BCR/ABL t(9;22) Dual-Color, Single-Fusion, Extra Signal Mm-BCR/ABL t(9;22) Dual-Color, Single-Fusion, Extra Signal Issue 4 BCR/ABL t(9;22) Dual-Color, Dual-Fusion - Cat# KBI-10005 is designed to detect the t(9;22)(q34;q11) reciprocal translocation. The der(9) and the der(22), the Ph chromosome will be observed as 2 yellow (red/green) fusion signals. This design will also detect cryptic insertions of ABL1 into the BCR region and therefore diagnosed as Ph-negative. A cryptic insertion of ABL1 in the BCR gene region will show a yellow (red/green) fusion signal and an additional small remaining red signal at the der(9). Normal Cell t(9;22) positive Cryptic insertion 9q34 to 22q11 D22S940 IGLC1 340 KB SHGC-147754 ASS 340 KB BCR ABL 1000 KB 1000 KB 22q11 NUP214 9q34 D9S1991 IGLL1 SHGC-107450 9 22 BCR/ABL t(9;22) Triple-Color, Dual-Fusion - Cat# KBI-10006 is designed from the same regions as the Dual-Color, Dual-Fusion probe but with the proximal BCR region labeled in blue. The probe is designed to detect both rearranged chromosomes. The der(22), which will be observed as purple (red/blue) fusion signal and der(9) which will show a yellow (red/green) fusion signal. Deletions of 9q or 22q can also be observed with this design. A deletion at the proximal 5’ site of ABL1 (9q34) will lead to lack of a red signal and a single green signal for 3’ distal sequences of the BCR gene region, deletions at the 3’ site of the BCR (22q11) gene will lead to lack of a green signal. Normal Cell t(9;22) positive t(9;22) positive with del(22q11) t(9;22) positive with del(9q34) D22S940 IGLC1 340 KB SHGC-147754 ASS 340 KB BCR ABL 1000 KB 1000 KB 22q11 NUP214 9q34 9 D9S1991 IGLL1 SHGC-107450 22 9 KREATECH NEWS BCR/ABL t(9;22,) Dual-Color, Single-Fusion, Extra Signal - Cat # KBI-10008 is designed to detect the t(9;22)(q34;q11) reciprocal translocation. By adding an additional region proximal to the breakpoints on chromosome 9q34, this probe will provide a smaller extra red signal at the der(9).The der(22) is visualized by a yellow (red/green) fusion signal. IGLC1 SHGC-147754 D22S940 ASS 340 KB 22q11.2 340 KB ABL 1000 KB Normal Cell t(9;22) positive 22 BCR NUP214 9q34 IGLL1 D9S1991 9 SHGC-107450 BCR/ABL t(9;22) Dual-Color, Single-Fusion - Cat# KBI-10009 is a simple design solely for the detection of the der(22), visible as one yellow (red/green) fusion signal while the der(9) will show no signal. IGLC1 SHGC-147754 D22S940 ASS 22q11.2 340 KB ABL 1000 KB Normal Cell t(9;22) positive 22 BCR NUP214 9q34 IGLL1 D9S1991 9 SHGC-107450 Mm-BCR/ABL t(9;22) Dual-Color, Single-Fusion, Extra Signal - Cat# KBI-10013 is designed to detect the der(22) with a break in the major breakpoint region (M-BCR) by one yellow (red/green) fusion signal. A smaller extra red signal will identify the der(9). Breaks in the minor breakpoint region (m-BCR) will be identified by two yellow (red/green) fusion signals. No smaller extra red signal should be visible. Normal Cell t(9;22) BCR/ABL with m-BCR t(9;22) BCR/ABL with M-BCR 1 IGLC1 m-BCR D22S940 SHGC-147754 ASS 22q11.2 420 KB 480 KB ABL 22 620 KB NUP214 9q34 9 10 D9S1991 HUMUT7039 BCR IGLL1 SHGC-107450 2 3 4 6 5 7 8 9 10 11 12 13 14 15 16 b1 b2 b3 b4 b5 M-BCR HUMUT7039 17 19 21 23 18 20 22 Issue 4 REFERENCES [1] Dewald et al., 1998, Blood 91: 3357-3365 [2] Huntly et al., 2003, Blood 102: 1160-1168 [3] Kolomietz et al., 2001, Blood 97: 3581-3588 [4] Mian et al., 2012, Haematol. 97: 251-257 [5] Sharma et al., 2010, Ann Hematol., 89: 241-7 [6] Tkachuk et al., 1990, Science 250: 559-562 To try our BCR/ABL probes please contact your local representative. ORDERING INFORMATION Probe name Tests Cat# BCR/ABL t(9;22) Dual-Color, Dual-Fusion 10 20 KBI-10005 KBI-12005 BCR/ABL t(9;22) Triple-Color, Dual-Fusion 10 KBI-10006 BCR/ABL t(9;22) Dual-Color, Single-Fusion, Extra Signal 10 KBI-10008 BCR/ABL t(9;22) Dual-Color, Single-Fusion 10 KBI-10009 Mm-BCR/ABL t(9;22) Dual-Color, Single-Fusion, Extra Signal 10 KBI-10013 11 KREATECH NEWS Development and validation of a REPEAT-FREE™ DNA-FISH assay to detect FGFR1 gene locus amplification Dimitri Pappaioannou1,3, Saskia Schoenmakers1, Birgit Rupp2, Isabell Dolznig2, Richard Ackbar2, Marcus Otte2, Sandor Snoeijers1 1 KREATECH Diagnostics, Vlierweg 20, 1032 LG, The Netherlands, 2 ORIDIS Biomarkers GmbH, Stiftingtalstrasse 5, A-8010 Graz, Austria, 3 Author for correspondence (d.pappaioannou@kreatech.com) * This article is edited from a paper that has been presented by the authors during the ADAPT (Accelerating Development & Advancing Personalized Therapy) meeting, September 19-21, Washington DC. INTRODUCTION METHOD Amplification of the fibroblast growth factor receptor type 1 gene (FGFR1) has been observed in numerous cancer types including Squamous Cell Carcinoma (SCC) Lung Cancer and Breast Cancer (BC)[1]. With the development of new therapeutic strategies, FGFR1 amplification might act as a valuable biomarker for R&D and can provide an attractive approach for clinical stratification[2]. PHASE 1 Design selection A series of probe designs specific for the FGFR1 gene locus (8p11) was constructed In Silico. Criteria for Probe design: • REPEAT-FREE™ design (no need for Cot-1 DNA) / In Silico Design • Free of segmental duplications in genomic regions covered • Optimize size of gene locus probe regions for desired signal intensity • Balance between signal strength of gene specific region probe and control probe Here we describe the development process of a REPEAT-FREE™ (RF) DNA-FISH assay tested for the detection of FGFR1 amplification. The process was divided into four phases: 1) design selection and assay development, 2) candidate selection, 3) batch testing of final design and, 4) validation on a cohort of 100 FFPE patient samples. Assay Development Candidate probes were tested on human cell lines (verified for FGFR1 amplification by real-time PCR). Using the REPEAT- FREE™-FGFR1/SE8 probe, FGFR1 gene locus amplification was considered positive with an FGFR/SE8 signal ratio ≥ 2.0. Polysomy of chromosome 8 was identified by an SE8/ nucleus ratio ≥ 2.0. PHASE 2 Candidate Selection From the initial probe designs, 2 designs were selected for further development. The probes were compared using various hybridization protocols and results between KREATECH and ORIDIS labs were compared as an indication of reproducibility. This resulted in selection ofthe final probe design (fig. 2). PHASE 3 Batch Testing Three batches of the final design were produced and tested for batch to batch TABLE 1. SCORING SCHEME FOR FGFR1 GENE PROBE COMPARISON ON TMA SECTIONS RECORDED FOR EACH PROBE BATCH 12 SCORE SAMPLE EVALUABILLITY SIGNAL INTENSITY (NUMBER OF CELLS) BACKGROUND SIGNAL INTENSITY PATTERN EVALUATION 1 > 40 Excellent None to minimal Correct FGFR1 pattern 2 ≤ 40 Good Acceptable Correct FGFR1 pattern 3 ≤ 25 Weak Disturbing Correct FGFR1 pattern 4 ≤ 10 Lack of signals Hampers analysis Wrong FGFR1 pattern Issue 4 variability on a TMA (core Ø = 0.6mm) according to criteria described in Table 1. PHASE 4 Validation on patient cohort Hybridization efficacy was tested on FFPE samples from 100 SCC NSCLC patients from 4 different sites in Europe and the US. Efficacy was determined by evaluation of signal intensity, background intensity and evaluability of sample and pattern. Repeated hybridizations were carried out to investigate the robustness and reproducibility of the assay. Evaluation was conducted by three independent observers. In addition, the assay was further tested on a cohort of BC samples. This study was approved by the local ethical review board of the Medical University of Graz. RESULTS PHASES 1 & 2 Design selection, Assay Development & Candidate Selection The final design of the REPEAT-FREE™ FGFR1 amplification probe is shown in figure 2. RH46977 8p11 SE8 FGFR1 540KB D8S414 8 Fig. 2. Graphic display of the final design of the REPEATFREE™ FGFR1 amplification probe (not to scale). A] SCC NSCLC C] unamplified B] BC D] amplified E] polysomy Fig. 3. Panels A] and B]: FGFR1 (8p11) / SE 8 probe hybridized to SCC NSCLC and BC tissue. Panels C], D] and E]: examples of FGFR1 copy numbers observed in tissue samples PHASE 3 Batch Testing Three production batches of FGFR1 probe were compared side by side and evaluated by 3 independent observers (see table 1 for evaluation criteria). Although variations occurred between observers, no variability between batches was observed (see table 2). In total 16 patient samples (8 NSCLC and 8 BC) and 12 cell lines were analyzed. FGFR1 gene status was known for all samples and this was confirmed for all three batches by all three observers. PHASE 4 Validation on Patient Cohort Finally, FGFR1 FISH was validated on TMAs containing a cohort of NSCLC samples (n = 100), selected for FGFR1 gene locus amplification, polysomy of chromosome 8, normal gene status and tissues lacking evaluable signal. Amplification of FGFR1, polysomy and normal gene status (fig. 3) were correctly identified for all samples. Furthermore evaluation was possible in 5/20 samples previously lacking detectable FISH signal with the optimized FGFR1 assay. CONCLUSIONS The REPEAT-FREE™-FGFR1 amplification assay allows clear detection of amplification and allows discrimination between amplification and polysomy, with high degrees of sensitivity, specificity and hybridization efficacy. The REPEAT-FREE™FGFR1 amplification assay fulfills all criteria for a clinical research tool aimed at patient stratification. The described assay development process is highly effective for probe design selection and testing of assay robustness and reproducibility and can be generally applied in the development of further FISH assays. TABLE 2. CONTINGENCY TABLE FOR EVALUATION OF INTENSITY COMPARED AT INDIVIDUAL SAMPLE LEVEL FOR ALLE THREE BATCHES OBSERVER 1 OBSERVER 1 OBSERVER 1 Batch 1 Score: 1 Score: 2 Score: 1 Batch 2 Score: 1 Score: 2 Score: 1 Batch 3 Score: 1 Score: 2 Score: 1 REFERENCES [1] Weiss et al., Sci. Transl. Med. 2(62): 62ra93 (2010) [2] Brooks et al., Clin. Can. Res. 18(7): 1855-62 (2012) 13 KREATECH NEWS ® THERMOBRITE ELITE AUTOMATED LABORATORY ASSISTANT AUTOMATED SAMPLE PREPARATION WITH REPEAT-FREETM POSEIDONTM FISH PROBES The ThermoBrite Elite provides total automation for the pre- and post-hybridization steps in FISH testing and provides on-board denaturation and hybridization. The ThermoBrite Elite can process up to 12 slides per run. You can also denature and hybridize slides on your ThermoBrite slide denaturation and hybridization system, in order to have the ThermoBrite Elite system available for additional runs. Perfect Solution for Pathology The pre-installed validated KREATECH FISH protocols allow for easy selection of the correct program for your solid tumor, bone marrow and blood samples. Just load your slides and walk away. Minimal hands-on time frees time for other important projects. Adding the probe and placing the coverslip are the only manual steps. After denaturation and hybridization, the system will complete the posthybridization steps. Just add the DAPI / antifade and coverslip and you are ready to image your slides. Interactive easy-to-use software The intuitive easy-to-use software allows you to run standard validated KREATECH protocols for blood, bone marrow and solid tumor FFPE samples. The system allows input of 10 different reagents. It has 3 independent waste ports. You can also control the functions in each of the 3 chambers (4 slides per chamber) allowing for small batches or running of 12 slides at one time. Features • • • • • 14 Automatedd fluidic system Accurate temperature control to + 1°C Controlledd agitation and mixing User-friendly dly Graphical User Interface Validated KREATECH protocols pre-installed Key Benefits • Improves consistency and reproducibility • Reduces hands-on time • Flexible and easy-to-use • Saves time and money Applications in FISH • Pathology (Solid tumor/ FFPE) • Hematology (Blood/bone marrow) • Cytology (Fluids) Issue 4 NOTES 15 KREATECH NEWS Events CONTACT The following events will be attended by members of KREATECH Diagnostics in the coming months. Please come and talk to us if you are at any of these events: 16 Nov Landelijke Analistendag Genoom Diagnostiek Media Plaza, Utrecht, The Netherlands 19 Nov – 23 Nov Carrefour Pathologie INTERNATIONAL KREATECH Diagnostics Vlierweg 20 1032 LG Amsterdam The Netherlands Phone: +31 (0)20 691 9181 Fax: +31 (0)20 630 4247 E-mail: customerservice@kreatech.com Paris, France BENELUX 2013 7 Jan Satellite Meeting: New techniques in Molecular Pathology Utrecht, The Netherlands 8 Jan – 9 Jan Winter Meeting Joint Meeting with the Dutch Pathological Society Utrecht, The Netherlands 18 Jan – 20 Jan 15. Bamberger Morphologietage 2013 Bamberg, Germany 27 Mar – 29 Mar Société Française d'Hématologie (SFH) Meeting CNIT Paris - La Défense, France KREATECH Diagnostics Vlierweg 20 1032 LG Amsterdam The Netherlands Phone: +31 (0)6 4850 0107 Fax: +31 (0)35 656 4826 E-mail: customerservice@kreatech.com France KREATECH Diagnostics 20 Avenue de la Paix 67080 Strasbourg Cedex France Phone: +33 (0)1 4372 0079 Fax: +33 (0)1 4348 8244 E-mail: customerservice@kreatech.com Germany KREATECH Diagnostics Vlierweg 20 1032 LG Amsterdam The Netherlands Phone: +49 (0)223 3713 5979 Fax: +31 (0)20 630 4247 E-mail: customerservice@kreatech.com For more information on events and for more KREATECH news please scan the QR code below or go to http://www.kreatech.com/news-media/events.html United Kingdom KREATECH Diagnostic 52 New Town, Uckfield East Sussex, TN22 5DE United Kingdom Phone: +44 (0)208 350 5430 Fax: +44 (0)208 711 3132 E-mail: customerservice@kreatech.com www.kreatech.com Disclaimer Marketing Materials: The content of this brochure is explicitly not meant for the North American region. If you are a resident in this region please contact the North American sales office to obtain the appropriate product information for your country of residence. For more information please visit our website: www.kreatech.com. 16 ©2012 KREATECH Diagnostics Published October 2012
© Copyright 2024