Intratumoral Patterns of Clonal Evolution in Meningiomas as Defined by Multicolor Interphase Fluorescence in Situ Hybridization (FISH)
2004; Elsevier BV; Volume: 6; Issue: 4 Linguagem: Inglês
10.1016/s1525-1578(10)60527-2
ISSN1943-7811
AutoresJosé María Sayagués, María Dolores Tabernero, A. Maíllo, Ana Marı́a Espinosa, Ana Rasillo, P. Díaz, Juana Ciudad, Antonio López, Marta Merino, J. Gonçalves, Ángel Santos‐Briz, Francisco Morales, Alberto Órfão,
Tópico(s)Glioma Diagnosis and Treatment
ResumoMeningiomas are cytogenetically heterogeneous tumors in which chromosome gains and losses frequently occur. Based on the intertumoral cytogenetic heterogeneity of meningiomas, hypothetical models of clonal evolution have been proposed in these tumors which have never been confirmed at the intratumoral cell level. The aim of this study was to establish the intratumoral patterns of clonal evolution associated with chromosomal instability in individual patients as a way to establish tumor progression pathways in meningiomas and their relationship with tumor histopathology and behavior. A total of 125 meningioma patients were analyzed at diagnosis. In all cases, multicolor interphase fluorescence in situ hybridization (iFISH) studies were performed on fresh tumor samples for the detection of quantitative abnormalities for 11 different chromosomes. In addition, overall tumor cell DNA content was measured in parallel by flow cytometry. iFISH studies were also performed in parallel on tissue sections in a subset of 30 patients. FISH studies showed that 56 (45%) of the 125 cases analyzed had a single tumor cell clone, all these cases corresponding to histologically benign grade I tumors. In the remaining cases (55%) more than one tumor cell clone was identified: two in 45 cases (36%), three in 19 (15%), and four or more clones in five cases (4%). Overall, flow cytometric analysis of cell DNA contents showed the presence of DNA aneuploidy in 44 of these cases (35%), 30% corresponding to DNA hyperdiploid and 5% to hypodiploid cases; from the DNA aneuploid cases, 35 (28%) showed two clones and 9 (7%) had three or more clones. A high degree of correlation (r ≥ 0.89; P < 0.001) was found between FISH and flow cytometry as regards the overall quantitative DNA changes detected with both techniques, the former being more sensitive. Among the cases with chromosome abnormalities, the earliest tumor cell clone observed was frequently characterized by the loss of one or more chromosomes (64% of all meningiomas); loss of either a single chromosome 22 or, less frequently, of a sex chromosome (X or Y) and del (1p) was commonly found as the single initial cytogenetic aberration (30%, 5%, and 5% of the cases, respectively). Interestingly, an isolated loss of chromosome 22 was only found as the initial abnormality in one out of 14 atypical/anaplastic meningiomas, while the same cytogenetic pattern was present in the ancestral tumor cell clone of 32% of the benign tumors. Cytogenetic patterns based on chromosome gains were found in the ancestral tumor cell clone in 4% of the patients, 2% corresponding to tetraploid tumors. Overall, cytogenetic evolution of the earliest tumor cell clones was frequently associated with tetraploidization (31%). Our results show that meningiomas are genetically heterogeneous tumors that display different patterns of numerical chromosome changes, with the presence of more than one tumor cell clone detected in almost half of the cases including all atypical/anaplastic cases. Interestingly, the pathways of intratumoral clonal evolution observed in the benign tumors were different from those observed in atypical/anaplastic meningiomas, suggesting that the latter tumors might not always represent a more advanced stage of histologically benign meningiomas. Meningiomas are cytogenetically heterogeneous tumors in which chromosome gains and losses frequently occur. Based on the intertumoral cytogenetic heterogeneity of meningiomas, hypothetical models of clonal evolution have been proposed in these tumors which have never been confirmed at the intratumoral cell level. The aim of this study was to establish the intratumoral patterns of clonal evolution associated with chromosomal instability in individual patients as a way to establish tumor progression pathways in meningiomas and their relationship with tumor histopathology and behavior. A total of 125 meningioma patients were analyzed at diagnosis. In all cases, multicolor interphase fluorescence in situ hybridization (iFISH) studies were performed on fresh tumor samples for the detection of quantitative abnormalities for 11 different chromosomes. In addition, overall tumor cell DNA content was measured in parallel by flow cytometry. iFISH studies were also performed in parallel on tissue sections in a subset of 30 patients. FISH studies showed that 56 (45%) of the 125 cases analyzed had a single tumor cell clone, all these cases corresponding to histologically benign grade I tumors. In the remaining cases (55%) more than one tumor cell clone was identified: two in 45 cases (36%), three in 19 (15%), and four or more clones in five cases (4%). Overall, flow cytometric analysis of cell DNA contents showed the presence of DNA aneuploidy in 44 of these cases (35%), 30% corresponding to DNA hyperdiploid and 5% to hypodiploid cases; from the DNA aneuploid cases, 35 (28%) showed two clones and 9 (7%) had three or more clones. A high degree of correlation (r ≥ 0.89; P < 0.001) was found between FISH and flow cytometry as regards the overall quantitative DNA changes detected with both techniques, the former being more sensitive. Among the cases with chromosome abnormalities, the earliest tumor cell clone observed was frequently characterized by the loss of one or more chromosomes (64% of all meningiomas); loss of either a single chromosome 22 or, less frequently, of a sex chromosome (X or Y) and del (1p) was commonly found as the single initial cytogenetic aberration (30%, 5%, and 5% of the cases, respectively). Interestingly, an isolated loss of chromosome 22 was only found as the initial abnormality in one out of 14 atypical/anaplastic meningiomas, while the same cytogenetic pattern was present in the ancestral tumor cell clone of 32% of the benign tumors. Cytogenetic patterns based on chromosome gains were found in the ancestral tumor cell clone in 4% of the patients, 2% corresponding to tetraploid tumors. Overall, cytogenetic evolution of the earliest tumor cell clones was frequently associated with tetraploidization (31%). Our results show that meningiomas are genetically heterogeneous tumors that display different patterns of numerical chromosome changes, with the presence of more than one tumor cell clone detected in almost half of the cases including all atypical/anaplastic cases. Interestingly, the pathways of intratumoral clonal evolution observed in the benign tumors were different from those observed in atypical/anaplastic meningiomas, suggesting that the latter tumors might not always represent a more advanced stage of histologically benign meningiomas. In recent years, an increasing number of studies have been reported which show that chromosome gains and losses are a frequent finding in meningioma tumors;1Zattara-Cannoni H Gambarelli D Dufour H Figarella D Vollot F Grisoli F Vagner-Capodano AM Contribution of cytogenetics and FISH in the diagnosis of meningiomas: a study of 189 tumors.Ann Genet. 1998; 41: 164-175PubMed Google Scholar, 2Weber RG Bostrom J Wolter M Baudis M Collins VP Reifenberger G Lichter P Analysis of genomic alterations in benign, atypical, and anaplastic meningiomas: toward a genetic model of meningioma progression.Proc Natl Acad Sci USA. 1997; 94: 14719-14724Crossref PubMed Scopus (352) Google Scholar, 3Ketter R Henn W Niedermayer I Steilen-Gimbel H Konig J Zang KD Steudel WI Predictive value of progression-associated chromosomal aberrations for the prognosis of meningiomas: a retrospective study of 198 cases.J Neurosurg. 2001; 95: 601-607Crossref PubMed Scopus (95) Google Scholar, 4Zang KD Meningioma: a cytogenetic model of a complex benign human tumor, including data on 394 karyotyped cases.Cytogenet Cell Genet. 2001; 93: 207-220Crossref PubMed Scopus (135) Google Scholar, 5Cai DX Banerjee R Scheithauer BW Lohse CM Kleinschmidt-Demasters BK Perry A Chromosome 1p and 14q FISH analysis in clinicopathologic subsets of meningioma: diagnostic and prognostic implications.J Neuropathol Exp Neurol. 2001; 60: 628-636PubMed Google Scholar, 6Sayagués JM Tabernero MD Maillo A Diaz P Rasillo A Bortoluci A Gomez-Moreta J Santos-Briz A Morales F Orfao A Incidence of numerical chromosome aberrations in meningioma tumors as revealed by fluorescence in situ hybridization (FISH) using 10 chromosome-specific probes.Cytometry. 2002; 50: 153-159Crossref PubMed Scopus (31) Google Scholar at the same time these studies have provided information on the specific cytogenetic abnormalities accumulated.1Zattara-Cannoni H Gambarelli D Dufour H Figarella D Vollot F Grisoli F Vagner-Capodano AM Contribution of cytogenetics and FISH in the diagnosis of meningiomas: a study of 189 tumors.Ann Genet. 1998; 41: 164-175PubMed Google Scholar, 2Weber RG Bostrom J Wolter M Baudis M Collins VP Reifenberger G Lichter P Analysis of genomic alterations in benign, atypical, and anaplastic meningiomas: toward a genetic model of meningioma progression.Proc Natl Acad Sci USA. 1997; 94: 14719-14724Crossref PubMed Scopus (352) Google Scholar, 3Ketter R Henn W Niedermayer I Steilen-Gimbel H Konig J Zang KD Steudel WI Predictive value of progression-associated chromosomal aberrations for the prognosis of meningiomas: a retrospective study of 198 cases.J Neurosurg. 2001; 95: 601-607Crossref PubMed Scopus (95) Google Scholar, 4Zang KD Meningioma: a cytogenetic model of a complex benign human tumor, including data on 394 karyotyped cases.Cytogenet Cell Genet. 2001; 93: 207-220Crossref PubMed Scopus (135) Google Scholar, 5Cai DX Banerjee R Scheithauer BW Lohse CM Kleinschmidt-Demasters BK Perry A Chromosome 1p and 14q FISH analysis in clinicopathologic subsets of meningioma: diagnostic and prognostic implications.J Neuropathol Exp Neurol. 2001; 60: 628-636PubMed Google Scholar, 6Sayagués JM Tabernero MD Maillo A Diaz P Rasillo A Bortoluci A Gomez-Moreta J Santos-Briz A Morales F Orfao A Incidence of numerical chromosome aberrations in meningioma tumors as revealed by fluorescence in situ hybridization (FISH) using 10 chromosome-specific probes.Cytometry. 2002; 50: 153-159Crossref PubMed Scopus (31) Google Scholar Of these, monosomy 22/22q−, and to a lesser extent 14q−, 1p− and 10q− abnormalities, together with loss of a sex chromosome (Y in males and X in females) and tetraploid karyotypes, are by far the most commonly observed aberrations. The analysis of large series of patients, using fluorescence in situ hybridization (FISH) techniques on interphase (iFISH) nuclei has provided a further accurate estimation of the incidence of these chromosomal abnormalities and their potential clinical significance.6Sayagués JM Tabernero MD Maillo A Diaz P Rasillo A Bortoluci A Gomez-Moreta J Santos-Briz A Morales F Orfao A Incidence of numerical chromosome aberrations in meningioma tumors as revealed by fluorescence in situ hybridization (FISH) using 10 chromosome-specific probes.Cytometry. 2002; 50: 153-159Crossref PubMed Scopus (31) Google Scholar, 7Maillo A Diaz P Sayagues JM Blanco A Tabernero MD Ciudad J Lopez A Goncalves JM Orfao A Gains of chromosome 22 by fluorescence in situ hybridization in the context of an hyperdiploid karyotype are associated with aggressive clinical features in meningioma patients.Cancer. 2001; 92: 377-385Crossref PubMed Scopus (31) Google Scholar, 8Maillo A Orfao A Sayagues JM Diaz P Caballero M Morales F Tabernero D A new classification scheme for the prognostic stratification of meningioma based on chromosome 14 abnormalities, patient's age, and tumor histopathology.J Clin Oncol. 2003; 21: 3285-3295Crossref PubMed Scopus (68) Google Scholar, 9Scholz M Gottschalk J Striepecke E Firsching R Harders A Fuzesi L Intratumorous heterogeneity of chromosome 10 and 17 in meningiomas using non-radioactive in situ hybridization.J Neurosurg Sci. 1996; 40: 17-23PubMed Google Scholar, 10Kasai H Kawamoto K Cytogenical analysis of brain tumors by FISH (fluorescence in situ hybridization) and FCM (flow cytometry).Noshuyo Byori. 1995; 12: 75-82PubMed Google Scholar, 11Lekanne Deprez RH Riegman PH van Drunen E Warringa UL Groen NA Stefanko SZ Koper JW Avezaat CJ Mulder PG Zwarthoff EC Cytogenetic, molecular genetic, and pathological analyses in 126 meningiomas.J Neuropathol Exp Neurol. 1995; 54: 224-235Crossref PubMed Scopus (96) Google Scholar For many years it has been well-established that the development of various human tumors including colorectal carcinomas,12Giaretti W A model of DNA aneuploidization and evolution in colorectal cancer.Lab Invest. 1994; 71: 904-910PubMed Google Scholar, 13Fearon ER Vogelstein B A genetic model for colorectal tumorigenesis.Cell. 1990; 61: 759-767Abstract Full Text PDF PubMed Scopus (10080) Google Scholar gliomas,14Collins VP James CD Gene and chromosomal alterations associated with the development of human gliomas.FASEB J. 1993; 7: 926-930PubMed Google Scholar, 15Louis DN Gusella JF A tiger behind many doors: multiple genetic pathways to malignant glioma.Trends Genet. 1995; 11: 412-415Abstract Full Text PDF PubMed Scopus (192) Google Scholar renal cell tumors,16Kovacs G Molecular cytogenetics of renal cell tumors.Adv Cancer Res. 1993; 62: 89-124Crossref PubMed Scopus (195) Google Scholar prostate cancer,17Visakorpi T Hyytinen E Koivisto P Tanner M Keinanen R Palmberg C Palotie A Tammela T Isola J Kallioniemi OP In vivo amplification of the androgen receptor gene and progression of human prostate cancer.Nat Genet. 1995; 9: 401-406Crossref PubMed Scopus (1246) Google Scholar and head and neck squamous cell carcinomas18Califano J van der Riet P Westra W Nawroz H Clayman G Piantadosi S Corio R Lee D Greenberg B Koch W Sidransky D Genetic progression model for head and neck cancer: implications for field cancerization.Cancer Res. 1996; 56: 2488-2492PubMed Google Scholar follows a multi-step pathway where an increasing number of genetic aberrations are accumulated due to genetic and/or chromosome instability. Typically, specific patterns of genetic evolution have been associated with both a more advanced stage and a more aggressive course of the disease.19Jin C Jin Y Wennerberg J Akervall J Dictor M Mertens F Karyotypic heterogeneity and clonal evolution in squamous cell carcinomas of the head and neck.Cancer Genet Cytogenet. 2002; 132: 85-96Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 20Garcia SB Novelli M Wright NA The clonal origin and clonal evolution of epithelial tumours.Int J Exp Pathol. 2000; 81: 89-116Crossref PubMed Scopus (57) Google Scholar, 21Fulci G Ishii N Maurici D Gernert KM Hainaut P Kaur B Van Meir EG Initiation of human astrocytoma by clonal evolution of cells with progressive loss of p53 functions in a patient with a 283H TP53 germ-line mutation: evidence for a precursor lesion.Cancer Res. 2002; 62: 2897-2905PubMed Google Scholar, 22Kattar MM Kupsky WJ Shimoyama RK Vo TD Olson MW Bargar GR Sarkar FH Clonal analysis of gliomas.Hum Pathol. 1997; 28: 1166-1179Abstract Full Text PDF PubMed Scopus (54) Google Scholar, 23Coons SW Johnson PC Shapiro JR Cytogenetic and flow cytometry DNA analysis of regional heterogeneity in a low grade human glioma.Cancer Res. 1995; 55: 1569-1577PubMed Google Scholar In a similar way, some models of clonal evolution have been proposed in meningiomas based on the intertumoral cytogenetic heterogeneity found, through the analysis of large series of patients by conventional karyotyping.4Zang KD Meningioma: a cytogenetic model of a complex benign human tumor, including data on 394 karyotyped cases.Cytogenet Cell Genet. 2001; 93: 207-220Crossref PubMed Scopus (135) Google Scholar These models provide hypothetical evolution pathways for what occurs during the sequential transition from normal meningeal cells to grade I, and grade II/grade III tumors.1Zattara-Cannoni H Gambarelli D Dufour H Figarella D Vollot F Grisoli F Vagner-Capodano AM Contribution of cytogenetics and FISH in the diagnosis of meningiomas: a study of 189 tumors.Ann Genet. 1998; 41: 164-175PubMed Google Scholar, 3Ketter R Henn W Niedermayer I Steilen-Gimbel H Konig J Zang KD Steudel WI Predictive value of progression-associated chromosomal aberrations for the prognosis of meningiomas: a retrospective study of 198 cases.J Neurosurg. 2001; 95: 601-607Crossref PubMed Scopus (95) Google Scholar, 11Lekanne Deprez RH Riegman PH van Drunen E Warringa UL Groen NA Stefanko SZ Koper JW Avezaat CJ Mulder PG Zwarthoff EC Cytogenetic, molecular genetic, and pathological analyses in 126 meningiomas.J Neuropathol Exp Neurol. 1995; 54: 224-235Crossref PubMed Scopus (96) Google Scholar However, so far such models have not been confirmed at the intratumoral level. This is probably related to the fact that conventional karyotyping techniques have several major limitations for the assessment of intratumoral clonal evolution in meningiomas: 1) they allow the analysis of only a small fraction of all tumor cells; 2) the use of cultured samples may induce selective growth of specific tumor cell clones; and 3) the low number of metaphases analyzed makes it difficult to accurately identify the different tumor cell clones present in a sample. In recent years, alternative cytogenetic techniques have been developed which facilitate the analysis of chromosome abnormalities in both interphase cells and metaphase chromosomes. Among others, these include iFISH and flow cytometry assessment of the DNA ploidy status of tumor cells. Although neither technique provides specific information on every chromosomal abnormality present in a tumor, they do allow a sensitive, rapid, and precise assessment of intratumoral heterogeneity. Multicolor iFISH is particularly suited to assessing intratumoral genetic heterogeneity, provided that adequate combinations of probes are used.24Rasillo A Tabernero D Sánchez ML Pérez de Andrés M Martín M Hernández J Moro MJ Fernández-Calvo J Sayagues JM Bortoluci A San Miguel J Orfao A Fluorescence in situ hybridization analysis of aneuploidization patterns in monoclonal gammopathy of undetermined significance versus multiple myeloma and plasma cell leukemia.Cancer. 2003; 97: 601-609Crossref PubMed Scopus (42) Google Scholar In the present study, we have applied multicolor iFISH analysis of 12 different probes specific for DNA sequences of 11 chromosomes in combination with flow cytometry DNA cell content measurements in a series of 125 consecutive patients, to explore the intratumoral cytogenetic heterogeneity of meningiomas. Our goal is to establish the intratumoral pathways of clonal evolution associated with chromosomal instability in individual meningioma patients as a way to identify more precisely the cytogenetic stage of each individual neoplasia and its relationship with the histopathology and behavior of the tumor. A total of 125 consecutive patients, 44 males and 81 females, diagnosed with meningioma at the Neurosurgical Unit of the University Hospital of Salamanca were analyzed. Mean age at diagnosis was 58 ± 15 years (range, 16 to 82 years). All cases were diagnosed with either intracranial (n = 118; 94%) or spinal (n = 7; 6%) meningiomas based on conventional histological criteria.25Louis DN, Scheithauer BW, Budka H, von Deimling A, Kepes JJ: Meningeal tumours. Kleihues P, Cavenee WK: World Heath Organization Classification of Tumours: Pathology and Genetics of Tumours of the Nervous System 2000:pp 176-184 LyonGoogle Scholar, 26Kleihues P Burger PC Scheithauer BW The new WHO classification of brain tumours.Brain Pathol. 1993; 3: 255-268Crossref PubMed Scopus (1481) Google Scholar Patient distribution according to the World Health Organization classification25Louis DN, Scheithauer BW, Budka H, von Deimling A, Kepes JJ: Meningeal tumours. Kleihues P, Cavenee WK: World Heath Organization Classification of Tumours: Pathology and Genetics of Tumours of the Nervous System 2000:pp 176-184 LyonGoogle Scholar, 26Kleihues P Burger PC Scheithauer BW The new WHO classification of brain tumours.Brain Pathol. 1993; 3: 255-268Crossref PubMed Scopus (1481) Google Scholar was as follows: 89% of the cases were grade I tumors; 10%, grade II; and the remaining 1%, grade III meningiomas. Tumor specimens were obtained by conventional surgical procedures and were then cut into several parts. Part of the tumor showing both macroscopic and microscopic infiltration was used to prepare single cell suspensions as described below. The remaining tumor was fixed in formalin (Paureac Quimicasa, Barcelona, Spain) and embedded in paraffin (Merck, Darmstadt, Germany). From these latter samples, sections were cut from three different areas representative of the tumoral tissue and placed over poly L-lysine coated slides (BioGenex, San Ramon, CA). All tissues were evaluated after hematoxylin-eosin (Merck) staining to confirm the presence and quantity of tumor cells infiltrating the material to be studied. Infiltration by tumor cells in tissue areas in the vicinity of those used for iFISH analysis of single cell suspensions was always 65%. Tumor cell DNA content studies were performed in all cases using fresh tumor samples obtained at diagnostic surgery. Single tumor cell suspensions were obtained by mechanical disaggregation and the cells' DNA was stained with propidium iodide (PI) (Sigma, St. Louis, MO) using techniques which have been previously described in detail.27Vindelov LL Christensen IJ Nissen NI A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis.Cytometry. 1983; 3: 323-327Crossref PubMed Scopus (1605) Google Scholar, 28Maillo A Diaz P Blanco A Lopez A Ciudad J Hernandez J Morales F Perez-Simon JA Orfao A Proportion of S-phase tumor cells measured by flow cytometry is an independent prognostic factor in meningioma tumors.Cytometry. 1999; 38: 118-123Crossref PubMed Scopus (18) Google Scholar Once sample preparation was completed and within a maximum period of 1 hour, DNA cell content measurements were performed in a FACSCalibur flow cytometer (Becton/Dickinson Biosciences (BDB), San Jose, CA). Information on a minimum of 104 tumor cells per sample was acquired using the CellQUEST software program (BDB). A second tube containing a 1/1 mixture of cells from the tumor sample and peripheral blood mononuclear cells from a gender-matched healthy volunteer was prepared and measured in parallel. DNA aneuploidy was defined as the presence of two distinct G0/G1 populations of cells with different DNA cell contents. The presence of more than one tumor cell clone by flow cytometry was defined by the observation of two or more G0/G1 populations of tumor cells. DNA index was calculated for each tumor cell clone as the ratio between the modal intensity of the PI-associated fluorescence of the G0/G1 tumor cells and that obtained for the normal G0/G1 diploid cells present in the same tube. The mean coefficient of variation for the G0/G1 peak of tumor cells was 3.3 ± 1%. In a subset of 22 meningiomas in which the diploid cells by iFISH could have derived from contamination by leukocytes, simultaneous staining with an anti-pan leukocyte antigen CD45 antibody conjugated with fluorescein isothiocyanate (FITC) (BDB) and the DRAQ5 DNA-dye (Biostatus, Cambridge, UK) was performed as previously described.29Smith PJ Wiltshire M Davies S Patterson LH Hoy T A novel cell permeant and far red-fluorescing DNA probe, DRAQ5, for blood cell discrimination by flow cytometry.J Immunol Methods. 1999; 229: 131-139Crossref PubMed Scopus (76) Google Scholar Multicolor interphase FISH (iFISH) studies were performed in all cases on an aliquot of the same single cell suspension prepared from the tumor sample and used for the flow cytometric measurement of cell DNA contents, after fixation in 3/1 methanol/acetic (v/v) (Merck). For the investigation of numerical chromosome abnormalities by iFISH, the following probes (Vysis Inc; Downers Grove, IL) specific for those chromosomes and chromosome regions more frequently gained or deleted in meningiomas6Sayagués JM Tabernero MD Maillo A Diaz P Rasillo A Bortoluci A Gomez-Moreta J Santos-Briz A Morales F Orfao A Incidence of numerical chromosome aberrations in meningioma tumors as revealed by fluorescence in situ hybridization (FISH) using 10 chromosome-specific probes.Cytometry. 2002; 50: 153-159Crossref PubMed Scopus (31) Google Scholar were systematically used in double-stainings: 1) for chromosomes 9 and 22: LSI BCR/ABL dual-color probe; 2) for chromosomes 15 and 17: LSI PML/RAR-α dual-color probe; 3) for chromosomes 14 and 18: LSI IgH/BCL2 dual-color probe; 4) for chromosomes X and Y: CEP X DNA probe, conjugated with Spectrum Orange (SO) and CEP Y DNA probe, conjugated with Spectrum Green (SG); 5) for chromosomes 7 and 10: CEP 7 DNA probe, conjugated with SO and CEP 10 DNA probe, conjugated with SG; and 6) for chromosome 1: CEP 1 DNA probe, conjugated with SO. In addition, the Midisatellite 1p36 probe directly labeled with FITC (D1, Q-BIOgene, Amsterdam, The Netherlands) was also used. To accurately define the exact abnormalities carried by a tumor cell clone within a tumor sample, further appropriate two-color stainings were performed, whenever necessary. Fixed cells were dropped into ethanol/ether (1/1, v/v) cleaned slides according to conventional cytogenetic protocols. The slides were then sequentially incubated with a solution containing 0.1 mg/ml pepsin (Sigma) (10 minutes at 37°C), fixed in 1% acid-free formaldehyde (Merck) (10 minutes at room temperature (RT)) and dehydrated in decreasing concentrations of ethanol (Merck) in water (100%, 95%, 70%). Once dried, the slides containing both the cells' DNA and the probes' DNA were denatured at 75°C (1 minute) and subsequently hybridized overnight at 37°C in a Hybrite thermocycler (Vysis, Inc.). After this incubation, slides were sequentially washed for 5 minutes at 46°C in 50% formamide (Merck) in 2X SSC (Merck) and then in 2X SSC. Afterward, cells were counter-stained with 35 μl of a mounting medium containing 75 ng/ml of DAPI (Sigma); Vectashield (Vector Laboratories Inc, Burlingame, CA) was used as anti-fading agent. In addition, FISH analysis was also performed on tissues sections from 30 tumors. Briefly, unstained 5-μm paraffin sections on electrostatically charged slides (BioGenex; San Ramon, CA) were baked overnight (12 hours to 24 hours) at 65°C and immersed in xylene (Merck) (3 × 15 minutes). Afterward, they were dehydrated in ethanol and washed with distilled water. The slides were pre-treated in a pressure cooker with 10 mmol/L citric acid-trisodium salt (Sigma) buffer (pH 6.0) for 4 minutes and washed with 2X SSC. For enzymatic digestion, incubation with proteinase K (Sigma) was performed for 15 minutes at 37°C. After washing with 2X SSC, the slides were fixed with 1% formaldehyde, washed with 2X SSC, and dehydrated with 70%, 90%, and 100% ethanol. The slides were hybridized in the Hybrite thermocycler using 10-μl probe/slide. Denaturation was performed at 90°C for 6 minutes and hybridized overnight at 38°C. Post-hybridization washes were carried out at 46°C with 50% formamide and counterstaining with DAPI was used, as described above. A BX60 fluorescence microscope (Olympus, Hamburg, Germany) equipped with a 100X oil objective was used for the enumeration of hybridization spots per nuclei. At least 200 nuclei were counted per slide. Only those spots with a similar size, intensity, and shape were counted in areas with <1% unhybridized cells; doublet signals were considered as single spots. A tumor was considered to carry a numerical abnormality for a given chromosome when the proportion of cells displaying an abnormal number of hybridization spots for the corresponding chromosome probe was at a percentage higher than the mean value + 2 SD of the percentage obtained with the same probe in control samples. The mean percentage of cells ± 1 SD showing one chromosome loss/gain in control samples was: chromosome 1p, 1.47 ± 1.22/0.14 ± 0.55; chromosome 1q, 0.47 ± 0.71/1.23 ± 1.30; chromosome 7, 0.33 ± 0.79/0.24 ± 0.46; chromosome 9, 1.47 ± 1.11/0.64 ± 0.7; chromosome 10, 1.04 ± 1.04/0.27 ± 0.50; chromosome 14, 1.53 ± 0.88/0.69 ± 1.11, chromosome 15, 1.09 ± 1.01/0.42 ± 0.68, chromosome 17, 1.84 ± 0.96/0.5 ± 0.85; chromosome 18, 0.38 ± 0.65/0.36 ± 0.99; chromosome 22, 1.73 ± 1.07/0.54 ± 0.85; chromosome X in females, 0.98 ± 0.88/0.13 ± 0.31; chromosome X in males, 0.1 ± 0/1 ± 0.87; and chromosome Y, 0.60 ± 1/1.08 ± 0.8. A tumor cell clone carrying a chromosomal abnormality was defined as being present when cells carrying identical numbers of hybridization spots for all of the probes analyzed, including the altered ones, were found at frequencies higher than 4% of the total cells analyzed in the sample. The expected amount of total mean DNA content per cell according to the relative amount of DNA abnormally gained or lost corresponding to each altered chromosome by FISH was calculated according to two different mathematical formulas: 1) chromosome index (CI): 1+ [(Ch1p × 0.022) − (2 × 0.022)] + [(Ch1q × 0.022) − (2 × 0.022)] + [(Ch7 × 0.027) − (2 × 0.027)] + [(Ch9 × 0.024) − (2 × 0.024)] + [(Ch10 × 0.023) − (2 × 0.023)] + [(Ch11 × 0.024) − (2 × 0.024)] + [(Ch14 × 0.018) − (2 × 0.018)] + [(Ch15 × 0.017) − (2 × 0.017)] + [(Ch17 × 0.015) − (2 × 0.015)] + [(Ch18 × 0.014) − (2 × 0.014)] + [(Ch22 × 0.009) − (2 × 0.009)] + [(Chx × 0.024) − (x × 0.024)] + [(Chy × 0.009) − (y × 0.009)] and 2) corrected chromosome index (CCI) = [(Ch1p × 0.022) + (Ch1q × 0.022) + (Ch7 × 0.027) + (Ch9 × 0.024) + (Ch10 × 0.023) + (Ch11 × 0.024) + (Ch14 × 0.018) + (Ch15 × 0.017) + (Ch17 × 0.015) + (Ch18 × 0.014)+ (Ch22 × 0.009) + (Chx × 0.024) + (Chy × 0.009)] ÷ [(2 × 0.043) + (2 × 0.027) + (2 × 0.024) + (2 × 0.023) + (2 × 0.024) + (2 × 0.018) + (2 × 0.017) + (2 × 0.015) + (2 × 0.014)+ (2 × 0.009) + (x × 0.027) + (y × 0.009)], where Chn is the number of spots per cell found for each probe; ch x was 2 in women and 1 in men and ch y was 0 in women and 1 in men. Tetraploid tumor cell clones were
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