Targeting multiple genetic aberrations in isolated tumor cells by spectral fluorescence in situ hybridization

Cancer Detection and Prevention 26 (2002) 171–179

Targeting multiple genetic aberrations in isolated tumor cells by spectral fluorescence in situ hybridization Marilyn L. Slovak, PhD a,c,∗ , Feiyu Zhang, MS a , Lucene Tcheurekdjian, BS a , Dolores Bobadilla, BS a , Victoria Bedell, BS a , Daniel A. Arber, MD b , Diane L. Persons, MD c , Jeffrey A. Sosman, MD d , Joyce L. Murata-Collins, PhD a a

Department of Cytogenetics, City of Hope National Medical Center, Room 2255, Northwest Building, 1500 East Duarte Road, Duarte, CA 91010, USA b Department of Anatomic Pathology, City of Hope National Medical Center, Duarte, CA 91010, USA c SWOG Cytogenetics Committee, Southwest Oncology Group (SWOG-9431), Operations Office, 14980 Omicron Drive, San Antonio, TX 78245, USA d Melanoma Committee, Southwest Oncology Group (SWOG-9431), Operations Office, 14980 Omicron Drive, San Antonio, TX 78245, USA Received 27 March 2002; received in revised form 2 May 2002; accepted 2 May 2002

Abstract Purpose: Tumorigenesis is characterized by the stepwise accumulation of multiple genetic changes that modify specific growth controls and cell survival. Conventional fluorescence in situ hybridization (FISH) assays reliably target one to three probes in a single hybridization. Simultaneous detection of more than three chromosomal or gene targets should increase the overall power of molecular cytogenetics by permitting detection of multiple genetic aberrations at the single cell level. Method: Spectral FISH (S-FISH) is an innovative molecular cytogenetic approach that can target many specific chromosomal aberrations in interphase and metaphase cells in a single hybridization, using combinatorial fluorescence and digital imaging microscopy. Results: S-FISH is a reliable means to identify disease-specific aberrations at the DNA level in individual tumor cells in hematopoietic disorders and malignant melanoma. Conclusion: S-FISH is a sensitive assay for the diagnosis and monitoring of disease-specific or patient-specific genetic aberrations, with significant clinical application in oncology for early detection of new or re-emerging abnormal clones, allowing for earlier therapeutic intervention. © 2002 International Society for Preventive Oncology. Published by Elsevier Science Ltd. All rights reserved. Keywords: FISH; Spectral FISH; Molecular cytogenetics; DNA; Minimal residual disease; Spectral imaging

1. Introduction Human cancers have been defined as clonal disorders arising from single cells that have accumulated multiple mutations either through inherited germline alterations or by acquired mutations of somatic cellular genes by environmental factors. Biologic and epidemiological evidence suggest that tumor formation is a consequence of multiple genetic hits resulting in dysregulation of genes affecting immortalization, transformation, cell cycle progression and checkpoint pathways, invasion/metastasis and angiogenesis. In both hematopoietic disorders and solid tumors, these genetic alterations are manifest as non-random chromosomal aberrations. Many of these aberrations are diagnostic and ∗ Corresponding author. Tel.: +1-626-359-8111x62313; fax: +1-626-301-8877. E-mail address: [email protected] (M.L. Slovak).

prognostic markers, e.g. chromosome region 9p21 has been observed in chromosomal inversions, translocations, heterozygous deletions and homozygous deletions in malignant cells from glioma, non-small cell lung cancer, leukemia and melanoma. This locus indicates the involvement of cyclin-dependent kinase inhibitor-2A (CDKN2A, also referred to as p16 or p16I NK4a ) genes in the genesis of several tumor types [1,2]. The rapid and reliable detection of critical genetic lesions in patient specimens can contribute to improved cancer risk assessment, prognostication of tumor behavior, and detection of minimal residual disease. Furthermore, the development of cancer drugs that target specific genetic abnormalities, such as HER2/neu gene amplification and Herceptin® therapy in breast cancer or t(9;22) translocations and Gleevac® therapy in chronic myeloid leukemia emphasizes the clinical value of defining appropriate molecular targets that direct novel therapeutic strategies [3].

0361-090X/02/$ – see front matter © 2002 International Society for Preventive Oncology. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 3 6 1 - 0 9 0 X ( 0 2 ) 0 0 0 6 3 - 6

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DNA-based molecular evaluation of chromosomal regions is known as fluorescence in situ hybridization (FISH). FISH assays permit reliable detection of one to three targets in a single hybridization. However, ploidy heterogeneity within a tumor could mask a specific gain or loss of a genetic locus unless a second (identifier) or several probes were used simultaneously. Simultaneous detection of multiple chromosomal or gene targets would relieve this concern and increase the overall information generated from FISH assays at the single cell level. Previously, we described the use of an innovative molecular cytogenetic approach termed spectral FISH or S-FISH that targeted multiple numeric chromosomal aberrations or aneuploidy in interphase nuclei in a single hybridization, using combinatorial fluorescence and digital imaging microscopy [4]. We now demonstrate the application of S-FISH using locus-specific FISH probe panels to survey for the presence or absence of recurring disease-specific aberrations in the hematopoietic disorders and malignant melanoma. The panels were selected to target locus-specific sites associated with tumor progression (i.e. molecular markers of metastatic melanoma) and the commonly observed non-random cytogenetic aberrations observed in therapy-related myelodysplasia/acute leukemia. The potential clinical utility of the latter to screen autologous stem cell aliquots prior to transplantation is evident.

2. Materials and methods 2.1. Classic and 24-color cytogenetics Cytogenetic preparations for hematopoietic and melanoma tumor samples were prepared using established methods. Spectral or 24-color karyotyping was performed using residual cell pellets stored at −80 ◦ C as described previously [5]. Cytogenetic nomenclature followed ISCN (1995) [6]. 2.2. S-FISH assay Residual cell pellets from cytogenetic samples stored at −80 ◦ C and touch preparations of malignant melanoma stored at −20 ◦ C were used for the S-FISH assay. Immediately prior to hybridization, cell pellets were washed in 3:1 methanol:glacial acetic acid (Carnoy’s fixative) solution and dropped onto pre-cleaned, non-silanized slides. Slides were pretreated in 2X SSC at 37 ◦ C for 30 min, dehydrated in an ethanol series (70, 80 and 95% for 2 min each), denatured in 70% formamide/2X SSC (pH 7.0) at 72 ◦ C for 2 min, and then dehydrated again in an ethanol series. Touch preparations of metastatic melanoma were made from tumors submitted to the SWOG Tumor Repository, Cincinnati, OH, according to Southwest Oncology Group (SWOG) protocol SWOG-9431. Touch preparations were made on silanized slides, fixed in ice cold Carnoy’s fixative and air-dried.

Table 1 Spectral FISH: t-MDS/AML DNA Probe

Label

Targets of panel 1 5q31 (EGR1)a 5p15 7q22 (CUTL1) 7␣-Satellite 8␣-Satellite

RPCI-1 98O22 RPCI-11 35K22 H-RG 305102 pZ7.5 pZ8.4

Spectrum orange Cy5.5 Texas red/Cy5.5 Texas red Spectrum green

Targets of panel 2 13q14 (RB1) 13qtel 17p13.1 (TP53) 17q12 (HER2/neu) 20q11.2 20p13

RPCI-11 174i10 RPCI-11 190i5 RPCI-11 199f11 RPCI-11 62n23 RPCI-1 81g23 RPCI-11 48M7

Cy5.5 Spectrum orange Texas red Cy5.5/spectrum orange Spectrum green Cy5.5/spectrum green

a Gene abbreviations: EGR1, early growth response 1; CUTL1, cut-like 1 or CDP/CCAAT displacement protein; RB1, retinoblastoma-1; TP53, tumor protein p53; HER2/neu, human EGF receptor/neuroblastoma-derived.

Samples were stored in a −20 ◦ C freezer until hybridization. 2.3. t-MDS/AML S-FISH probe panel DNA probes that targeted non-random chromosomal aberrations frequently reported for t-MDS/AML, namely, −5/del(5q), −7/del(7q), +8/del(13q), del(17p) and del(20q) were obtained. Eleven probes localized to six chromosomes were used (Table 1). Probes for chromosomes 5, 13, 17 and 20 were obtained from Dr. Pieter J. de Jong, Children’s Hospital Oakland Research Institute (CHORI). Probes for the ␣-satellite regions of chromosome 8 (pZ8.4) and chromosome 7 (pZ7.5) were generous gifts from Dr. Mariano Rocchi, University of Bari, Italy. The 7q22 DNA probe (H-RG 305102) localized immediately telomeric to CUTL1 was a gift from Dr. Stephen W. Scherer, Hospital for Sick Children, Toronto, Ont., Canada. The malignant melanoma FISH probes were specifically chosen based on previous studies implicating their potential role in development or progression of malignant melanoma [7–12]. In this study, FISH probes for six different target genes/sites (LIBC, MART1, EGFR, and enumeration probes for chromosomes 6, 7 and 9) were used to identify gene copy number abnormalities in fresh tumor and touch preparations of metastatic melanoma (Table 2). Probes targeting 6q22 (RPCI-11 28K19) and 9ptel (RPCI-11 147N16 including the MART1 or melanoma antigen recognized by T cells 1 gene) were obtained from Dr. Pieter J. de Jong at CHORI. Probes for the ␣-satellite regions of chromosome 6 (pEDZ6), chromosome 7 (pZ7.5) and chromosome 9 (pMR9A) were gifts from Dr. Mariano Rocchi, University of Bari, Italy. California Institute of Technology provided the probe targeting the EGFR gene at 7p12.3 (2026N22). Probe DNAs for the hematopoietic and melanoma samples were isolated using the Qiafilter plasmid midi kit (Qiagen,

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3. Results

Table 2 Spectral FISH: melanoma DNA probe panel Targets (LIBC)a

6q22 6␣-Satellite 7p12.3 (EGFR) 7␣-Satellite 9ptel (MART1) 9␣-Satellite

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Probe

Label

RPCI-11 28K19 PEDZ6 2026N22 PZ7.5 RPCI-11 147N16 PMR9A

Cy5.5/spectrum green Spectrum green Texas red/Cy5.5 Texas red Cy5.5 Spectrum orange

a Gene abbreviations: LIBC, lost in inflammatory breast cancer; EGFR, epidermal growth factor receptor; MART1, melanoma antigen recognized by T cells 1.

Valencia, CA) and labeled by nick translation using standard technique. Probe combinatorial labeling, spectral imaging and image acquisition were performed as described [4]. Briefly, S-FISH combinatorial labeling strategy used four fluorochromes: spectrum orange, Texas red, spectrum green and Cy5.5. After the concentrations of each probe in the S-FISH panels were adjusted for equivalent fluorescence intensities, the desired probe cocktails were combined and unincorporated nucleotides removed using the QIAquick Nucleotide Removal Kit (Qiagen, Valencia, CA). Hybridization followed standard FISH procedures for locus-specific probes. Biotin-labeled probes were detected with 2.5 ␮g/ml Cy5.5 conjugated avidin (Rockland, Gilbertsville, PA) in 1X PBS, 1% blocking reagent (Roche Molecular Biochemicals, Indianapolis, IN) and 1% BSA for 5 min. Nuclei were counterstained with 4 ,6 -diamindino-2-phenylindole (DAPI). Spectral images were acquired and analyzed with the SD200 SpectraCube system (Applied Spectral Imaging Ltd., Migdal Haemek, Israel) attached to a Zeiss Axioplan II microscope with a 150 W xenon UV light source. System calibration and scoring criteria followed described procedures [4,13]. Validation of S-FISH was performed by classic cytogenetics and 24-color spectral karyotyping when metaphase cells were available or by conventional FISH analyses. 2.4. Single and dual color FISH Conventional FISH analyses were performed to confirm the chromosomal location of each probe prior to initiation of the analysis, to rule out cross hybridization, and to corroborate discrepant results. In addition to the probes mentioned earlier, P1 1069 (gift from Dr. Alexander Kamb, Myriad Genetics Inc., Salt Lake City, UT) which contains the p16I NK4a /MTS/CDKN2A gene, was used to test for 9p interstitial deletions in melanoma. Slide pretreatment was done as previously described for S-FISH assays. Hybridization followed standard procedure [14]. Biotin-labeled probes were detected with FITC conjugated avidin (Roche Molecular Biochemicals, Indianapolis, IN) in 1X PBS, 1% blocking reagent and 1% BSA for 5 min. Nuclei were counterstained with DAPI. Lymphocytes from a normal male were used as a negative control.

3.1. Secondary AML A bone marrow sample collected from a 71-year-old male with a diagnosis of acute myeloid leukemia evolving from myelodysplastic syndrome (secondary AML) was chosen for a comprehensive molecular cytogenetic work-up because of its karyotypic complexity and the unclear relationship between the observed aberrant hypodiploid and hyperdiploid clones. All mitotic cells collected for GTG-band analysis were clonally abnormal. Clone 1 was characterized by deletion of 5q, additional material of unknown origin translocated to 11p, a ring chromosome 11, a suspected paracentric inversion of 12p, deletion of 18q and losses of chromosomes 17 and 20. The karyotypic designation of hypodiploid clone 1 was: 44,XY,del(5)(q22q3?1),add(11)(p15),r(11)(p1?5q2?5),?inv (12)(p11.2p13.3),−17,del(18)(q2?2),−20 [cp13] (Fig. 1A). Clone 2 was hyperdiploid, with gains of chromosomes 2, 4, 6, 8, 9, 11, 13 and 21, losses of chromosomes 18 and 20, an augmentation of 5q and smaller derivative chromosome 5s. One hypertetraploid cell containing a duplicate copy of clone 2 aberrations was also collected. Losses and gains among the hyperdiploid cells resulted in the use of a composite karyotype with 51–56 chromosomes. The karyotypic designation of clone 2 was 51–56,XY,+2,+4,add(5)(q?11.2)x2,+add(5)(q3?5),+6,+8, +8,+9,+11,+13,−18,−20,+21 [cp6] (Fig. 1B). Twenty-four-color karyotyping was performed to refine analysis of the karyotypic aberrations observed in this case and possibly to define a more robust relationship between the two clones. In clone 1, the five aberrations were revised as follows: del(5)(q22q3?1) to der(5)t(5;?17;19),add(11)(p15) to der(11)t(5;11),?inv(12)(p11.2p13.3) to der(12)t(12;18), a normal 16 to der(16)t(1;16) and del(18q) to der(18)t(16;18). Because combinatorial fluorophore blending may occur at the site of translocation, the questionable presence of chromosome 17 material as part of a three-way translocation could not be established with the assay. In clone 2, the chromosome 5 aberrations were reassigned as der(5)t(5;17)x2, der(5)t(5;19) with insertion of chromosome 5 material into a derivative chromosome 10 and two apparently normal copies of chromosome 17. Tetrasomy 8 was confirmed in clone 2 only. Monosomy 20 was observed in both clones. Spectral FISH employed DNA FISH probe panels designed to screen for recurring cytogenetic aberrations in the myelodysplastic syndromes and acute myeloid leukemias including −5/del(5q), −7/del(7q), +8, del(13q), abnormalities of chromosome 17 including deletions and iso-chromosomes and 20q deletions (Table 1). Because two clones were present, metaphase cells were collected to view the genes of interest by location and copy number (Fig. 2A and B). In clone 1, the first S-FISH panel identified disomy for chromosomes 7 and 8 with deletion of EGR1 (early growth response 1) gene located at 5q31 in

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Fig. 1. Cytogenetic analysis of sAML: (panel A) hypodiploid clone (clone 1). ISCN designation is 44,XY,del(5)(q22q3?1),add(11)(p15), r(11)(p1?5q2?5),?inv(12)(p11.2p13.3),−17,del(18)(q2?2),−20 [cp13]. The 24-color karyotyping refined the karyotypic aberrations as follows: del(5)(q22q3?1) to der(5)t(5;?17;19),add(11)(p15) to der(11)t(5;11),?inv(12)(p11.2p13.3) to der(12)t(12;18), a normal 16 to der(16)t(1;16) and del(18q) to der(18)t(16;18); (panel B) hyperdiploid clone (clone 2). The karyotypic designation for clone 2 was 51–56,XY,+2,+4,add(5)(q?11.2)x2,+ add(5)(q3?5),+6,+8,+8,+9,+11,+13,−18,−20,+21 [cp6]. This cell contains 54 chromosomes with random loss of chromosomes 11 and 13. The chromosome 5 aberrations were reassigned after 24-color karyotyping as der(5)(5;17)x2,der(5)t(5;19) and ins(10;5). Tetrasomy 8 and two apparently normal copies of chromosome 17 were confirmed. Arrows point to clonal abnormalities. Monosomy 20 was observed in both clones.

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Fig. 2. S-FISH in sAML: (panel A) in clone 1, S-FISH panel A identified disomy for chromosomes 7 and 8 with deletion of EGR1/5q31 in one chromosome 5; (panel B) the second panel of probes revealed monosomy 20 with loss of one TP53/17p13 signal consistent with loss of one copy of chromosome 17 identified by cytogenetics. Two HER2/neu/17q12 signals were observed with the second signal localized to the der(5), confirming a complex (three-way) unbalanced 5;17;19 translocation as suggested by 24-color analysis (arrow); (panel C) in the hyperdiploid clone 2, S-FISH revealed three 5p15 signals, one 5q31 signal, disomy 7 and tetrasomy 8. The der(5)t(5;19) did not appear to have chromosome 17 material but 17q12 material was translocated to the two smaller derivative chromosome 5s (arrows); (panel D) S-FISH showed one TP53/17p13 signal with four HER2/neu/17q12 signals. The 17q12 signals were localized to the der(5)t(5;17)s, a normal chromosome 17 and an apparently del(17p), the latter not recognized as a deletion by either classic or 24-color karyotyping.

one chromosome 5. The second panel revealed monosomy 20, loss of one TP53 (17p13) signal consistent with the loss of chromosome 17 identified by cytogenetics. However, two HER2/neu/17q12 signals were observed with the second signal localized to the der(5), confirming a complex (three-way) unbalanced 5;17;19 translocation as suggested

by 24-color analysis. In clone 2, the der(5)t(5;19) did not appear to have 17 material, although two smaller derivative chromosome 5s did have distal 17q12, der(5)t(5;17). S-FISH showed only one signal for TP53/17p13 but four HER2/neu/17q12 signals with the two remaining 17q12 signals, one localized to a normal chromosome 17 and

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Fig. 3. Conventional FISH studies: (A and B) in the sAML case, dual color FISH using the 5p15 probe labeled in spectrum green and HER2/neu/17q12 probe labeled spectrum orange confirmed the der(5) rearrangements with chromosome 17 in clone 1 (A) and clone 2 (B). Arrows point to the der(5)s. (C) Dual color FISH using the MART1/9ptel probe labeled in spectrum orange and the CDKN2A/p16/9p21 probe label in spectrum green confirms loss of one copy of p16 gene in melanoma sample 165797.

Fig. 4. Malignant melanoma molecular cytogenetics: (A) 24-color karyotyping of SWOG-9431 malignant melanoma sample, 165797. The karyotypic designation for this near-diploid tumor was 48,X,−X,+der(1)t(9;X;1)(p13;p11.?4;q10),t(5;16)(q31.1;q13),der(6)t(1;6)(q10;p10),+der(6)t(6;22) (p10;q10),+7,del(9)(p22p24),der(9)t(X;9)(p11.?4;p13),der(13;15)(q10;q10),t(14;19)(q11.2;p13.3),+19,+20,−22[20]. This cell has random loss of chromosomes 12, 19 and 20. Three clonal 9p aberrations were identified. Arrows point to clonal aberrations involving chromosomes 6, 7 and 9; (B–E) spectral FISH of a malignant melanoma interphase cell from case 165797. (B) Spectral image of the interphase nucleus shown in panels C and D. (C) Classified image of same cell shown in panels B and D. (D) DAPI image of the cell. (E) Spectral FISH profile shows concordance with the 24-color karyotyping observed in panel A. Note the presence of three der(6) chromosomes with loss of 6q illustrated by loss of two copies of the LIBC gene, trisomy 7 and two MART1/9centromere signals.

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the other on an unrecognized del(17p) (Fig. 2C and D). Dual color FISH using the 5p15/17q12 probe combination confirmed der(5) rearrangements with chromosome 17 for both clones (Fig. 3A and B). The chromosome 5 material inserted into 10q was not composed of EGR1 or 5p15. 3.2. Malignant melanoma Cytogenetic and molecular cytogenetic studies were initiated on SWOG-9431 malignant melanoma samples. Fresh tumor material was used for classic and 24-color cytogenetic studies and touch preparations were prepared from frozen tumor material for S-FISH. For panel validation, a near diploid tumor was chosen for S-FISH evaluation. The 25 mitotic cells collected for GTG-band analysis and 24-color karyotyping from short-term tissue culture were clonally abnormal. An hyperdiploid stemline was characterized by gain of a derivative chromosome 1 resulting from a complex translocation involving Xp and 9p, a translocation between 5q and 16q, a derivative chromosome 6 resulting from a whole-arm translocation with 1q, gain of a derivative chromosome 6 resulting from a whole-arm translocation with 22q, deletion of 9p, a derivative chromosome 9 resulting from a translocation with Xp, a Robertsonian t(13;15), a translocation between proximal 14q and distal 19p, gains of additional copies of chromosomes 7, 19 and 20 and losses of X and 22 (Fig. 4). The final karyotypic designation was 48, X,-X,+der(1)t(9;X;1)(p13;p11.?4;q10),t(5;16)(q31.1;q13), der(6)t(1;6)(q10;p10), + der(6)t(6;22)(p10;q10), + 7,del(9) (p22p24),der(9)t(X;9)(p11.?4;p13),der(13;15)(q10;q10),t(14; 19)(q11.2;p13.3),+19,+20,−22[20]. S-FISH confirmed the presence of three derivative 6 chromosomes with loss of the LIBC/6q22 chromosomal region in two copies, trisomy 7, and two copies (disomy) for chromosome 9 and the 9p telomeric region containing the MART1 gene. Because three 9p clonal aberrations were apparent by cytogenetics, dual color FISH tested for the possibility of p16INK4a deletions. Loss of one p16/9p21 signal with two copies of MART1/9ptel in this melanoma case, indicated translocation of MART1 to chromosome 1 and an interstitial 9p deletion (Fig. 3C).

4. Discussion Molecular cytogenetics and FISH-based assays can contribute to improve cancer risk assessment. Previously, we reported the value of S-FISH for the assessment of minimal residual disease (MRD) by simultaneous detection of multiple numeric aberrations (aneuploidy) in a single hybridization [4]. S-FISH probe panels have been redesigned to detect disease-specific chromosomal targets; the simultaneous use of selected DNA probes provides internal confirmation of suspected losses or gains, increases the information gained from each assay, and most importantly, increases the clinical utility of the assay.

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Significant levels of karyotypically aberrant cells have been reported by conventional FISH in pre-transplant samples taken from patients who developed t-MDS/AML post-transplant for non-Hodgkin’s lymphoma [15,16]. One of our goals was to develop a DNA probe panel for the common t-MDS/AML aberrations. The intended application is to screen apheresis samples prior to autologous hematopoietic stem cell transplantation, particularly, in cases where the transplant candidates have received high doses of prior cytotoxic therapy. In this study, S-FISH applying DNA probes targeting 11 chromosome sites on six chromosomes was used to examine a karyotypically complex sAML case. It enabled definition of the poorly understood relationship between two vastly different clones and clarified the nature of aberrations involving chromosomes that are commonly reported in therapy-related MDS/AML. Although 24-color karyotyping defined two chromosome 5 aberrations in clone 1 as der(5)t(5;?17;19) and der(11)t(5;11), the questionable presence of chromosome 17 material as part of a three-way translocation in the der(5)t(5;?17;19) could not be established by this method. The combinatorial labeling profiles of these chromosomes varied by only one fluorochrome and blending of fluorescence emission spectra characteristic of each individual chromosome occurs with chromosomal rearrangements. As a result, the faint hint of an additional hybridization band at the translocation junction required confirmation using DNA region-specific probes. In this case, S-FISH and dual color FISH confirmed the presence of chromosome 17 material, and in addition, S-FISH identified the translocated chromosome 17 segment as 17q12 containing the HER2/neu gene, as part of a complex (three-way) unbalanced t(5;17;19)(q22;q12;?). The relationship between the hypodiploid and hyperdiploid clones in this sAML case was obscured by differing clone-specific aberrations. We also note that massive hyperdiploidy is an unusual finding in sAML [17,18]. With 24-color karyotyping, we identified four aberrations in clone 2 with chromosome 5 material, der(5)t(5;19), two copies of der(5)t(5;17) and an insertion of chromosome 5 material into 10q. In comparison there were only two chromosome 5 aberrations in clone 1, der(5)t(5;17;19) and der(11)t(5;11). In clone 2, S-FISH revealed one signal for 17p13 indicating a small submicroscopic deletion in one apparently normal chromosome 17, resulting in loss of a TP53 gene, despite the presence of four HER2/neu/17q12 signals, corroborating the 24-color karyotype. Comparison of the two clones revealed loss of 5q chromosomal material, loss of TP53, translocation of chromosome 17q12 material to 5q and monosomy 20 as common aberrations between them. Of interest, at follow-up 1 month later, the hyperdiploid clone was no longer evident and the hypodiploid clone showed additional evidence of clonal evolution. These observations reinforce the impression that vast genetic instability may be associated with an evolving acute leukemia arising from myelodysplasia in association with loss of TP53. Importantly for future studies, probe selection for detection

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of chromosome 5, 17 and 20 aberrations in t-MDS/AML appears to be suitable for further analyses. The SWOG Melanoma Biologics and Cytogenetics Committees recently initiated pilot cytogenetic and S-FISH studies on a subgroup of SWOG-9431 malignant melanoma tumors. As illustrated in our case, melanoma karyotypes are typically highly aneuploid with multiple numerical and structural aberrations, often involving chromosomes 1, 6, 7 and 9. Structural aberrations of chromosome 1 are found in ∼60% and rearrangements of chromosome 6 are found in nearly 80% of abnormal cases, frequently resulting in 6q loss [19]. Abnormalities of chromosome 9 can be numerical or structural, with deletion of 9p21 sequences particularly frequent. The 9p21 contains the p16INK4a locus, a site known to be critical for normal progression through the cell cycle [2]. Trisomy 7 is another recurrent finding in melanoma; implying that additional copies of genes on this chromosome including EGFR may play a role in melanoma development or tumor progression [20]. The S-FISH DNA probe panel for melanoma was selected based on a literature search of genes involved in melanoma development and progression. In preliminary analyses, the S-FISH assay proved reliable in the detection of loss of 6q22 and trisomy 7, with retention of two signals for MART1/9ptel and chromosome 9 centromere. However, the complexity of the genetic aberrations found in metastatic melanoma underscores the need to define reliable genetic markers to improve prognostication and identify new treatments for primary and advanced stage melanoma. We plan to refine a panel of DNA FISH probes that identify alterations in genes in malignant melanoma cells, based on their role in growth regulation, apoptosis, invasion/metastases, angiogenesis, and DNA repair. We expect that specific alterations in gene copy or the pattern of alterations observed in metastatic disease will serve as prognostic markers for progression of primary stage melanoma. Additionally, some genes (i.e. EGFR, C-KIT) may be identified as molecular targets to pursue with novel treatments, such as specific tyrosine kinase inhibitors, monoclonal antibodies or ligands capable of binding to cell surface receptors for growth factors in either advanced or early stage of disease. Despite the improved genetic diagnostic accuracy with S-FISH, we are aware of the need to overcome some technical challenges. The three dimensional aspects of interphase nuclei are problematic for FISH-based assays. In S-FISH this difficulty is amplified because the multiplicity of signals prohibits an accurate quantitative evaluation of overlapping signals, as well as the capturing of signals at different focal planes. These obstacles, and the labor intensity of combinatorial labeling and screening, indicate the needs for improved probe labeling, Z-stacking software, and automated spot counting. The incorporation of Z-stacking software and motorized microscopy should allow for automation in the near future. It would permit evaluation of fluorescence intensity of probe signals measured through different planes in the sample to produce a sharply detailed three-dimensional composite image of a single cell in two dimensions.

In summary, advances in molecular oncology and genomics will increasingly impact the clinical arena. New gene targets will be developed for use as diagnostic markers, prognostic risk factors, and potential sites for directed therapeutic intervention. Our preliminary studies with S-FISH suggest that this assay may contribute substantially to determination of tumor subtype, detection of residual disease, disease progression and, perhaps in the near future, response to treatment.

Acknowledgements The authors wish to thank Dr. Sandra Wolman for her critical review of this manuscript. This work was supported in part by NIH Grants CA30206, CA32102 and a private donation from the Bernard Ruttenberg family.

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