Please contact us or your local distributor

for the previous issue of KREATECH NEWS

You can also visit our website:

www.kreatech.comwww.kreatech.c

Please contact us or your local distributor for the previous issue

of KREATECH NEWS and for the HEMATOLOGY Brochure.

You can also visit our website: www.kreatech.com.

KREATECH DIAGNOSTICS

HEMATOLOGY DNA PROBES

RF™ POSEIDON™

SOLUTIONS FOR HEMATOLOGY FISH DIAGNOSTICS

Please contact us or your local distributor for

our Pathology Brochure and Catalogue 2011-2012.

You can also visit our website: www.kreatech.com

KREATECH DIAGNOSTICSSOLID TUMORS

RF™ POSEIDON™ SOLUTIONS FOR SOLID TUMOR FISH DIAGNOSTICS

© 2011 KREATECH D

iagnostics Publish

ed Januari 2011

KREATECH DIAGNOSTICS

CATALOGUE 2011 - 2012

KREATECH DIAGNOSTICSCATALOGUE 2011 - 2012

KREATECHNEWS

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.

We are proud to present a selection of informative articles from opinion leaders in 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

All prostate cancers become essentially within a few years resistant to endocrine therapy. This stage of disease is known as Castration-Resistant 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.

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

We are very grateful to these authors for their contribution to this newsletter and we hope that you will enjoy it.

Yours sincerely, Kreatech Diagnostics

IN THIS ISSUE

ALK REARRANGEMENT IN LUNG CANCER

ANDROGEN RECEPTOR IN PROSTATE CANCER

MLL TRANSLOCATIONS IN AML

Issue 1

KREATECH

NEWS

IN THIS ISSUE

FISH IN BIOCHIPS

IT TAKES TWO

FISH4U

Issue 2

This issue will start with an article that has been kindly contributed

by the group of Prof. dr. Jean-Louis Viovy from Laboratoire de Physico-

Chimie Curie, Institut Curie, Paris in France. This article describes the

miniaturization of FISH protocols using monolithic chips. To showcase

the possibilities of miniaturization of FISH protocols using monolithic

chips the group of professor Viovy have developed a procedure for

the molecular typing of HER2 using the SK-BR-3 cell line as a model.

The results show that such a microfluidic FISH platform opens new

possibilities for routine early-stage cancer screening on circulating cells.

Following this article you can read more on the use of the ULS™

labeling technology for comparative genomic hybridization by

arrayGCH and the application of the REPEAT-FREE™ technology for

subsequent confirmatory FISH.

WELCOME TO THE LATEST EDITION

OF THE KREATECH NEWSLETTER!

On page 7 of this newsletter you can read more about our FISH4U

program. Since the launch of this program we have received many

requests for the development of customized probes and in this

short article you can find more about how this program works and

what it can do for you.

Happy reading!

Sincerely, The KREATECH Diagnostics team

FISH4U BRAF-KIAA1549 (7q34) Triple-Color, Fusion Probe hybridized to pediatric glioma tissue showing fusion of BRAF-KIAA1549 by tandem duplication

(2R2G1F3B).

comcom

prevvioousus s ii

hure..

. SOLUTIONS

Please contact us ore contact us or your

our Pathology Brochure and Catalogue 2011gue 2011our Patholog -2012. -2012

You can also visit our website: www.kreatechw.kreatechYou .com.co

™ POSEIDON™ UTIONSNN FOR S SOLID TUMOR II FISH DIAGNOII SOO TICSCC

RF™

SOLU

© 2011 KREATECH D

iagnostics Publish

ed Januari 2011

KREATECH DIAGNOSTICSCATAA AT LOGUE 2011 - 2012

THIS ISSUE

SH IN BIOCHIPS

T TAKES TWO

ISH4U

uted

ico- the

case

lithic

re for

model.

s new

g cells.

ULS™

ion by

ogy for

IONONON IOIOIIOONI NOR!R!R!R!!

On page 7 of

program. Si

requests fo

short artic

what it c

Happy

Sincere

diatric glioma tissue sho

Please contact us or your local distributor for the previous issues of KREATECH NEWS

You can also visit our website: www.kreatech.com.www.kreatech.c

Please contact us or your local distributor for our Pathology Brochure and Catalogue 2011-2012. You can also visit our website: www.kreatech.com

KREATECH DIAGNOSTICSSOLID TUMORS

RF™ POSEIDON™ SOLUTIONS FOR SOLID TUMOR FISH DIAGNOSTICS

© 2011 KREATECH Dia

gnostics Pub lished J

anuari 2011

KREATECH DIAGNOSTICSCATALOGUE 2011 - 2012

K REATECH DI AGNOSTICSCATALOGUE 2011 - 2012

KREATECHNEWS

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.

We are proud to present a selection of informative articles from opinion leaders in 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

All prostate cancers become essentially within a few years resistant to endocrine therapy. This stage of disease is known as Castration-Resistant 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.

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

We are very grateful to these authors for their contribution to this newsletter and we hope that you will enjoy it.

Yours sincerely, Kreatech Diagnostics

IN THIS ISSUE

ALK REARRANGEMENT IN LUNG CANCERANDROGEN RECEPTOR IN PROSTATE CANCERMLL TRANSLOCATIONS IN AML

Issue 1

comcom.

12.

m

™ POSEIDON™ UTIONSNN FOR S SOLID TUMOR II FISH DIAGNOFIFI SOO TICSCC

RF™

SOL

© 2011 KREATECHDia

gnosticsPublishedJ

anuari 2011

CH DI AGNOSTICSCATAATLOGUE 2011 - 2012

Dear Reader,

Welcome to our latest edition issue we present our latest rFISH DNA probes and our Fprovides you with the probe

We are proud to present opinion leaders in Europe

Promising results have bCrizotinib (PF-0234106(NSCLC) patients carryIn Situ Hybridization the diagnosis of ALKPathology Departme

All prostate canceto endocrine therResistant ProstatCRPC cases the m(AR) overexpreRead more on Cancer by Pro

Translocatipartner gein Acute influenceeffect dexactlydecisio

We anew

Yo

N THIS ISSUE

ALK REARRANGEMENT NT T INN LANDROGEN RECEPTORR INN ORMLL TRANSLOCATIOONNS INNS IN

Please contact us or your local distributor for the previous issue

of KREATECH NEWS and for the HEMATOLOGY Brochure.

You can also visit our website: www.kreatech.com.

KREATECH DIAGNOSTICSHEMATOLOGY DNA PROBES

RF™ POSEIDON™ SOLUTIONS FOR HEMATOLOGY FISH DIAGNOSTICS

Please contact us or your local distributor for

our Pathology Brochure and Catalogue 2011-2012.

You can also visit our website: www.kreatech.com KREATECH DIAGNOSTICSSOLID TUMORS

RF™ POSEIDON™ SOLUTIONS FOR SOLID TUMOR FISH DIAGNOSTICS

RF™ PSOLOO UT

© 2 01 1 K RE A TE C H Di ag n os ti cs P u bl ish e d Ja nu a ri 20 1 1

KREATECH DIAGNOSTICSCATALOGUE 2011 - 2012

KREATECH DIAGNOSTICSCATALOGUE 2011 - 2012

E-mail: customerservice@kreatech.comWebsite: www.kreatech.comail: customerservice@kreatech. comte: www.kreatech.comomerservice@kreatech.comw.kreatech.comrvice@kreatech.comech.com

KREATECHNEWS

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.

We are proud to present a selection of informative articles from

opinion leaders in 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

All prostate cancers become essentially within a few years resistant

to endocrine therapy. This stage of disease is known as Castration-

Resistant 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.

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 7We are very grateful to these authors for their contribution to this

newsletter and we hope that you will enjoy it.

Yours sincerely, Kreatech Diagnostics

IN THIS ISSUEALK REARRANGEMENT IN LUNG CANCER

ANDROGEN RECEPTOR IN PROSTATE CANCER

MLL TRANSLOCATIONS IN AML

Issue 1

KREATECHNEWS IN THIS ISSUEFISH IN BIOCHIPSIT TAKES TWOFISH4U

Issue 2

This issue will start with an article that has been kindly contributed

by the group of Prof. dr. Jean-Louis Viovy from Laboratoire de Physico-

Chimie Curie, Institut Curie, Paris in France. This article describes the

miniaturization of FISH protocols using monolithic chips. To showcase

the possibilities of miniaturization of FISH protocols using monolithic

chips the group of professor Viovy have developed a procedure for

the molecular typing of HER2 using the SK-BR-3 cell line as a model.

The results show that such a microfluidic FISH platform opens new

possibilities for routine early-stage cancer screening on circulating cells.

Following this article you can read more on the use of the ULS™

labeling technology for comparative genomic hybridization by

arrayGCH and the application of the REPEAT-FREE™ technology for

subsequent confirmatory FISH.

WELCOME TO THE LATEST EDITION

OF THE KREATECH NEWSLETTER!

On page 7 of this newsletter you can read more about our FISH4U

program. Since the launch of this program we have received many

requests for the development of customized probes and in this

short article you can find more about how this program works and

what it can do for you.

Happy reading!Sincerely, The KREATECH Diagnostics team

FISH4U BRAF-KIAA1549 (7q34) Triple-Color, Fusion Probe hybridized to pediatric glioma tissue showing fusion of BRAF-KIAA1549 by tandem duplication

(2R2G1F3B).

IN THIS ISSUE…

GATA 4 (8p23) MICRODELETION IN CONGENITAL HEART DEFECT & DIAPHRAGMATIC HERNIABy Prof. Damien Sanlaville, MD, PhD, Laboratoire de CytogénétiqueConstitutionnelle, CHU de Lyon, France.

Patients with 8p23 microdeletion exhibit mild facial dysmorphism including micro-cephaly, enophtalmia, micrognathia, mode-rate intellectual deficiency, behaviour problems (they display sudden and extreme changes of behaviour, with outbursts of aggressiveness and destructive behaviour) and malformations. Two malformations are frequently associated with 8p23 micro-deletion, namely congenital heart defect (CHD) and congenital diaphragmatic hernia (CDH) [1]. The first description of a person with an 8p23 deletion was in 1988. To date more than 50 cases of interstitial or terminal 8p23.1 deletions have been reported. 8p23 deletions occur in equal frequency in males and females and across all ethnic groups. The exact frequency of this microdeletion is not known although is has been reported that it varies between 1/55.000 and 1/7000 live births [2]]... Read more on page 4

DETECTION OF PROGNOSTICALLY RELEVANT GENETIC ABNORMALITIES IN CHILDHOOD B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKAEMIA

By Prof. Christine J Harrison, Ms. Claire Schwab, Dr. Amy Erhorn.Leukaemia Research Cytogenetics Group,

Newcastle University, Newcastle-upon-Tyne, UKThe abnormalities with the most significant impact on treatment and management of childhood B-lineage acute lymphoblastic leukaemia are t(4;11)(q21;q23)/MLL-AFF1, t(9;22)(q34;q11)/BCR-ABL1 and near-haploidy/low hypodiploidy for high risk stratification…... Read more on page 2

KREATECH NEWS

Issue 3

OTHER NEWSCHRONIC LYMPHOCYTIC LEUKEMIA FISH ANALYSIS USING REPEAT-FREE™ POSEIDON™ DNA PROBES. Read more on page 8

ANNOUNCING THE THERMOBRITE® ELITE; AUTOMATION FOR FISH TESTING.Read more on page 10

FLUORESCENCE IN SITU HYBRIDISATION TO REFINE THE DIAGNOSIS OF BONE AND SOFT TISSUE TUMOURS

By Karoly Szuhai MD PhD1, Hans J Tanke PhD1 and Pancras C.W. Hogendoorn MD, PhD2.

1Department of Molecular Cell Biology and 2Department of Pathology, Leiden University

Medical Center, Leiden, The Netherlands. Primary bone and soft tissue tumours are a heterogeneous group of tumours including benign and malignant lesions. They have a mesenchymal origin, sometimes hypothesised to originate from mesenchymal stem cells [1]. The diagnosis of these lesions may be challenging in daily clinicopathological practice, as they are relatively rare: sarcomas, malignant mesenchymal tumours, are less than 2% of all cancers, 100 distinct subtypes are defined [2] Furthermore morphologic criteria of malignancy used for other cancer types are not always applicable [2] For instance, histologically “ominous” looking lesions may be benign (i.e. nodular fasciitis or ancient schwannoma) and deceptively “bland” lesions may have a malignant behaviour (i.e. low grade fibromyxoid sarcoma or epithelioid sarcoma) [2].… Read more on page 5

IN THIS ISSUE…

P53 / MPO “ISO 17Q” FISH PROBE IN CHRONIC LYMPHOCYTIC LEUKEMIA ROUTINE DIAGNOSTICS

Lana Harder, MD, PhDInstitute of Tumour Genetics North, Kiel, Germany

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 syn-dromes [4] and in up to 40% of complex aberrant acute myeloid leukemias [5]. In addition, P53 aberrations are recurrent ab-normalities in almost all solid and hema-tologic cancers [6].Read more on page 2

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. Griffi nUniversity of Kent, Kreatech, Digital Scientifi c 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

FGFR1 (8p11) / SE 8 probe hybridized to SCC NSCLC and BC tissue

KREATECH NEWS

Issue 4

Read more on page 5

OTHER NEWSREPEAT-FREE™ POSEIDON™ BCR/ABL1 t(9;22) PRODUCT RANGE Read more on page 8

DEVELOPMENT AND VALIDATION OF A REPEAT-FREE™ DNA-FISH ASSAY TO DETECT FGFR1 GENE LOCUS AMPLIFICATIONRead more on page 12

DETECTION OF NUP98 GENE REARRANGEMENTS BY FLUORESCENT IN SITU HYBRIDIZATION

Susana Lisboa, Manuel R. Teixeira Department of Genetics, Portuguese Oncology Institute, Porto, Portugal

Nucleoporin 98 gene (NUP98) rearrangements have been identifi ed 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

2

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 identifi ed prior to treatment [9, 11]. Cytogenetic analyses and fl uorescence 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].

DESIGN OF P53 FISH PROBESThe 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 specifi c 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.

P53 / MPO “ISO 17Q” FISH PROBE IN CHRONIC

LYMPHOCYTIC LEUKEMIA ROUTINE DIAGNOSTICS

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, PhDInstitute of Tumour Genetics North, Kiel, Germany

KREATECH NEWS

3

Issue 4

FISH PROBE EVALUATIONEvery 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 fi ve 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 signifi cance 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 specifi c copy number alterations, monosomal karyotype, and dismal outcome. Blood 119:211421-21.[6] Swerdlow SH. et al. 2008, WHO classifi cation 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.

[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: fi nal 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).

17p13

P53

330 KB

D17S960

D17S634

17

400 KBMPO17q22

SHGC-144222

D17S2151

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

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).

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).

To try out our P53 probes please contact your local representative

4

KREATECH NEWS

Nucleoporin 98 gene (NUP98) rearrangements have been identifi ed 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 fl anking 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 fi xed 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 humidifi ed 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 fl uorescence microscope. For each sample, 100 intact non-overlapping nuclei were scored. We have detected a normal signal pattern (two fusion signals) in all the

DETECTION OF NUP98 GENE REARRANGEMENTS

BY FLUORESCENT IN SITU HYBRIDIZATION

Susana Lisboa, Manuel R. Teixeira Department of Genetics, Portuguese Oncology Institute, Porto, Portugal

530 KB

NUP98

11p15

410 KB

RH75370

SHGC-79113

D11S4525

SHGC-84145

GAP 455 KB

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 fl uorescence 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).

5

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. Griffi nUniversity of Kent, Kreatech, Digital Scientifi c and London Bridge Fertility, Gynaecology and Genetics Centre.

INTRODUCTIONIn the May issue of Chromosome Research this year, Dimitris Ioannou and colleagues from Darren Griffi n’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 fl uorescence in situ hybridization (FISH) probes, on human in vitro fertilization (IVF) embryos (Ioannou et al 2012). Darren Griffi n was in fact involved in the fi rst 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 scientifi c 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 microarray-based 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 “follow-up” 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.

FLUOROCHROMELAYER A:

CENTROMERICPROBES

LAYER B:

CENTROMERIC PROBES

LAYER C:

CENTROMERIC PROBES

LAYER D: UNIQUE

SEQUENCE PROBES

PlatinumBright™ 405Dark blue SE7 (7p11-q11) SE11 (11p11-q11) SE18 (18p11-q11) CD37 (19q13)

PlatinumBright™ 415Light blue(aqua) SE1 (1q12) SE9 (9q12) SE16 (16p11-q11) PDGFRB (5q33)

PlatinumBright™ 495Green SE6 (6p11-q11) SE20 (20p11-q11) SE2 (2p11-q11) DSCR (21q22)

PlatinumBright™ 547Light red/orange SE8 (8p11-q11) SE12 (12p11-q11) SEX (Xp11-q11) BCR (22q11)

PlatinumBright™ 590Dark red SE3 (3p11-q11) SE10 (10p11-q11) SEY (Yp11-q11) RB (13q14)

PlatinumBright™ 647Far red SE4 (4p11-q11) SE17 (17p11-q11) SE15 (15p11-q11) IGH (14q32)

The fl uorescent 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

TABLE 1 THE PROBES USED AND THEIR LOCI IN EACH OF THE MULTI-COLOUR PROBE MIXES

6

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 fl uorochrome-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 fi rstly to confi rm 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 fi nally to assay the nuclear positions of the loci recognized by the Kreatech probes.

MATERIALS AND METHODSMaterial 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 fl uorochromes, 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 fi xed 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) fi x 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 pre-denature 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 fi rst three rounds of hybridization (centromeric probes) was for 30 min, whereas for the fi nal 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 fi nal rounds with the following exceptions: The overnight hybridization time for the fi nal round (previously mentioned), pepsin and paraformaldehyde treatment were only required for the fi rst round; the post-hybridization wash time was reduced with every round from 90 s (fi rst round of hybridization) to 50–60 s (second round) to 30 s (third and fi nal rounds). Microscopy analysis was performed on an Olympus BX-61 epifl uorescence microscope equipped with a cooled CCD camera (by Digital Scientifi c—Hamamatsu Orca-ER C4742-80) and using the appropriate fi lters. To enable analysis of the fl uorochromes for image acquisition two communicating fi lter wheels (Digital Scientifi c UK) with the appropriate fi lters were used. The recommended fi lters by the probe manufacturer can be found here: http://www.kreatech.com/rest/customer-service-support/technical-support/fl uorophores-and-fi lter-recommendation.html and the image capture system was SmartCapture (Digital Scientifi c UK).

Fig.1Blastomere nucleus after four rounds of hybridisation, scales bars=10μm. a DAPI only, showing retention of nuclear structure b Same nucleus with fi nal 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 fl uorochrome 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.

7

Issue 4

RESULTS AND DISCUSSIONAnalysis 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 confi rmation of the fi rst 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 confi rmed, 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 fi nancially not feasible given the numbers of nuclei that need to be analysed. The added benefi t 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, Griffi n 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, Griffi n 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 specifi c 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 specifi c 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

8

KREATECH NEWS

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 (fi gure 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.

22

BCR 22q11

9

der(9)

ABL 9q34

BCR-ABL1

ABL1-BCRA

der(22)

Figure1: BCR/ABL1 t(9;22) Dual-Fusion Assay

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 fi rst 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 confi rm 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.

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 specifi c 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).

The REPEAT-FREE POSEIDON portfolio contains fi ve 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, Extra Signal

BCR/ABL t(9;22) Dual-Color, Single-Fusion Mm-BCR/ABL t(9;22) Dual-Color, Single-Fusion, Extra Signal

9

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

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)

9q34

9

ASS 340 KB

NUP214

ABL

1000 KB

D9S1991

SHGC-147754

22q11

22

340 KB

IGLL1

BCR

1000 KB

D22S940

SHGC-107450

IGLC1

9q34

9

ASS 340 KB

NUP214

ABL

1000 KB

D9S1991

SHGC-147754

22q11

22

340 KB

IGLL1

BCR

1000 KB

D22S940

SHGC-107450

IGLC1

10

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.

Normal Cell t(9;22) positive

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.

Normal Cell t(9;22) positive

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 identifi ed 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

9q34

9

ASS 340 KB

NUP214

ABL

1000 KB

D9S1991

SHGC-147754

9q34

9

ASS

NUP214

ABL

1000 KB

D9S1991

SHGC-147754

9q34

9

ASS 420 KB

NUP214

ABL

620 KB

D9S1991

SHGC-14775422q11.2

22

480 KB

IGLL1

BCR

D22S940

SHGC-107450

IGLC1

1

234

6

89111315

17

19

21

23

1

234

6

89

1315

5

7

101214

16

17

19

21

18

20

22

23

b2b4

b1b3b5

m-BCR

M-BCR

HUMUT7039

HUMUT7039

22q11.2

22

340 KB

IGLL1

BCR

D22S940

SHGC-107450

IGLC1

22q11.2

22

340 KB

IGLL1

BCR

D22S940

SHGC-107450

IGLC1

11

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

ORDERING INFORMATION

Probe name Tests Cat#

BCR/ABL t(9;22) Dual-Color, Dual-Fusion1020

KBI-10005KBI-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

To try our BCR/ABL probes please contact your local representative.

12

KREATECH NEWS

INTRODUCTIONAmplification 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 amplifi cation might act as a valuable biomarker for R&D and can provide an attractive approach for clinical stratifi cation[2].

Here we describe the development process of a REPEAT-FREE™ (RF) DNA-FISH assay tested for the detection of FGFR1 amplifi cation. The process was divided into four phases: 1) design selection and assay development, 2) candidate selection, 3) batch testing of fi nal design and, 4) validation on a cohort of 100 FFPE patient samples.

METHODPHASE 1Design selectionA series of probe designs specifi c 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

specifi c region probe and control probe

Assay DevelopmentCandidate probes were tested on human cell lines (verifi ed for FGFR1 amplifi cation by real-time PCR). Using the REPEAT-

FREE™-FGFR1/SE8 probe, FGFR1 gene locus amplifi cation was considered positive with an FGFR/SE8 signal ratio ≥ 2.0. Polysomy of chromosome 8 was identifi ed by an SE8/nucleus ratio ≥ 2.0.

PHASE 2Candidate SelectionFrom 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 fi nal probe design (fi g. 2).

PHASE 3Batch TestingThree batches of the fi nal design were produced and tested for batch to batch

Development and validation of a REPEAT-FREE™ DNA-FISH

assay to detect FGFR1 gene locus amplifi cation

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.

SCORESAMPLE EVALUABILLITY

(NUMBER OF CELLS)SIGNAL INTENSITY

BACKGROUND SIGNAL

INTENSITYPATTERN 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

TABLE 1. SCORING SCHEME FOR FGFR1 GENE PROBE COMPARISON ON TMA SECTIONS RECORDED FOR EACH PROBE BATCH

13

Issue 4

variability on a TMA (core Ø = 0.6mm) according to criteria described in Table 1.

PHASE 4Validation on patient cohortHybridization effi cacy was tested on FFPE samples from 100 SCC NSCLC patients from 4 different sites in Europe and the US. Effi cacy 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.

RESULTSPHASES 1 & 2 Design selection, Assay Development & Candidate SelectionThe fi nal design of the REPEAT-FREE™ FGFR1 amplifi cation probe is shown in fi gure 2.

540KBFGFR1

8p11

RH46977

D8S414

8

SE8

Fig. 2. Graphic display of the fi nal design of the REPEAT-

FREE™ FGFR1 amplifi cation probe (not to scale).

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

TABLE 2. CONTINGENCY TABLE FOR EVALUATION OF INTENSITY

COMPARED AT INDIVIDUAL SAMPLE LEVEL FOR ALLE THREE BATCHES

A] SCC NSCLC

C] unamplifi ed D] amplifi ed E] polysomy

B] BC

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 3Batch TestingThree 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 confi rmed for all three batches by all three observers.

PHASE 4Validation on Patient CohortFinally, FGFR1 FISH was validated on TMAs containing a cohort of NSCLC samples (n = 100), selected for FGFR1 gene locus amplifi cation, polysomy of chromosome 8, normal gene status and tissues lacking evaluable signal. Amplifi cation of FGFR1,

polysomy and normal gene status (fi g. 3) were correctly identifi ed for all samples. Furthermore evaluation was possible in 5/20 samples previously lacking detectable FISH signal with the optimized FGFR1 assay.

CONCLUSIONSThe REPEAT-FREE™-FGFR1 amplifi cation assay allows clear detection of amplifi cation and allows discrimination between amplification and polysomy, with high degrees of sensitivity, specificity and hybridization effi cacy. The REPEAT-FREE™-FGFR1 amplification assay fulfills all criteria for a clinical research tool aimed at patient stratifi cation. 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.

REFERENCES [1] Weiss et al., Sci. Transl. Med. 2(62):

62ra93 (2010)[2] Brooks et al., Clin. Can. Res. 18(7):

1855-62 (2012)

14

KREATECH NEWS

THERMOBRITE® ELITEAUTOMATED LABORATORY ASSISTANT

The ThermoBrite Elite provides total automation for the pre- and post-hybridization steps in FISH testing and provides on-board denaturation and hybridization.

Perfect Solution for PathologyThe 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 post-hybridization steps. Just add the DAPI / antifade and coverslip and you are ready to image your slides.

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.

Interactive easy-to-use softwareThe 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• Automated fl uidic system• Accurate temperature control to + 1°C• Controlled agitation and mixing• User-friendly Graphical User Interface• Validated KREATECH protocols

pre-installed

Key Benefi ts• 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)

AUTOMATED SAMPLE PREPARATION WITH REPEAT-FREETM

POSEIDONTM FISH PROBES

d fl uidic systemtemperature control to + 1°Cd agitation and mixingdly Graphical User Interface

Key Benefi ts• Improves consistency and

reproducibility• Reduces hands-on time

Applications in FISH• Pathology (Solid tumor/

15

Issue 4

NOTES

16

CONTACT

INTERNATIONALKREATECH Diagnostics Vlierweg 201032 LG AmsterdamThe NetherlandsPhone: +31 (0)20 691 9181Fax: +31 (0)20 630 4247E-mail: customerservice@kreatech.com

BENELUXKREATECH Diagnostics Vlierweg 201032 LG AmsterdamThe NetherlandsPhone: +31 (0)6 4850 0107Fax: +31 (0)35 656 4826E-mail: customerservice@kreatech.com

FranceKREATECH Diagnostics20 Avenue de la Paix67080 Strasbourg CedexFrancePhone: +33 (0)1 4372 0079Fax: +33 (0)1 4348 8244E-mail: customerservice@kreatech.com

GermanyKREATECH Diagnostics Vlierweg 201032 LG AmsterdamThe NetherlandsPhone: +49 (0)223 3713 5979Fax: +31 (0)20 630 4247E-mail: customerservice@kreatech.com

United KingdomKREATECH Diagnostic52 New Town, Uckfi eldEast Sussex, TN22 5DEUnited KingdomPhone: +44 (0)208 350 5430Fax: +44 (0)208 711 3132E-mail: customerservice@kreatech.com

www.kreatech.com

©2012 KREATECH DiagnosticsPublished October 2012

KREATECH NEWS

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

Events

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

Paris, France

2013

7 Jan Satellite Meeting: New techniques in Molecular Pathology

Utrecht, The Netherlands

8 Jan – 9 Jan Winter MeetingJoint 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

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 offi ce to obtain the appropriate product information for your country of residence. For more information please visit our website: www.kreatech.com.