Peripheral Blood Samples
Sixty-four peripheral blood samples (5 ml, each) obtained from four groups of consenting individuals were investigated: (i) 20 from normal, cancer-free individuals, used as controls (hereafter termed CON); (ii) 20 from patients with hematological malignancies taken at diagnosis (hereafter termed ADI); (iii) 14 of patients with hematological malignancies obtained prior to allogeneic stem cell transplantation (alloSCT) (hereafter termed PRE); and (iv) 10 from patients with hematological malignancies taken following alloSCT, each displaying 100% donor chimerism, as judged from analyses of bone-marrow aspirates (hereafter termed POST). None of the investigated individuals was included in more than one group. The subjects of each group were chosen at random in order to avoid any bias in patients' selection.
The patients suffered from various hematological malignancies. Of the 20 ADI individuals 9 had acute myelogenous leukemia (AML), 8 chronic myelogenous leukemia (CML), 2 acute lymphoblastic leukemia (ALL), and 1 multiple myeloma (MM). Of the 14 PRE individuals, 4 had AML (3 in remission and one not), 8 CML (6 in the chronic and 2 in the accelerated phase), 1 ALL (in remission) and 1 non-Hodgkins lymphoma (NHL, not in remission). Of the POST subjects, 4 had AML, 4 had CML, 1 ALL and 1 NHL (all at full remission).
The ADI group included 9 females and 11 males, ranging in age from 3 to 80 years (median and mean values of 35.0 and 37.2 years, respectively). The PRE group contained 5 females and 9 males, ranging from 11 to 56 years (median and mean of 37.5 and 37.0 years, respectively). The POST group included 3 females and 7 males, ranging from 13-51 years (median and mean of 32.0 and 32.3 years, respectively). The CON group consisted of 8 females and 12 males, ranging from 11-65 years (median and mean of 36.0 and 35.0 years, respectively).
In addition, we examined 14 peripheral blood samples (5 ml each) obtained from 14 consenting urological patients, referred to prostate biopsy because of suspected prostate cancer. This group of male patients ranged in age 53-78 years, with median and mean values of 66.5 and 65.1 years, respectively. Following biopsy, four out of the 14 patients were diagnosed with prostate cancer and the rest (10 individuals) were found to be cancer-free. Of the 10 cancer-free individuals three were found to suffer from chronic prostate inflammation and seven from benign prostate hyperplasia.
Bone Marrow Samples
Twenty-six bone marrow samples, obtained from consenting subjects (2 ml each), were also tested. They included: (i) 12 exemplars from a new group of cancer-free individuals, 4 females and 8 males, ranging in age from 7-54 years; and (ii) 14 from patients in the PRE group, one sample from each of them.
Cell Cultures
Cell cultures were set up according to the standard protocol for karyotype analysis [18]. Briefly, aliquots of peripheral blood or bone marrow aspirates were introduced into F-10 medium supplemented with 20% fetal calf serum, 0.2% heparin and 1% penicillin/streptomycin antibiotic solution (Biological Industries, Israel). For blood cell culturing, this medium was additionally supplemented by 3% phytohemagglutinin (PHA) to stimulate cell division. Each of the blood samples derived from the CON, ADI and PRE patients were incubated in duplicate, one in the enhanced F-10 medium containing PHA and the second in a medium containing 10-7 M 5-azacytidine (AZA; Sigma, USA) as well as PHA.
After 72 hours at 37°C, colchicine (Sigma, USA) was added (to a final concentration of 5 × 10-7 M) for one hr, followed by a hypotonic treatment (0.075 M KCl at 37°C, for 15 min) and four washes, each with a fresh, cold (-20°C) 3:1 methanol:acetic acid solution. The cell suspensions were stored at -20°C until used for FISH.
Slide Preparation
The stored cell suspensions prepared for hybridization were washed twice in a 3:1 methanol: acetic acid solution, diluted until the suspension became slightly cloudy and then approximately 5 μl of the suspension was dropped onto the marked circles of two-well slide glasses. The two-well slides were obtained from Insitus Biotechnologies (Albuquerque, NM, USA) and used without any pretreatment.
Probes
We tested four loci using directly labeled commercial probes obtained from Vysis: (i) the TP53 probe (32-190006); (ii) the AML1 probe (LSI 21; 32-190002); (iii) the α-satellite probe specific for centromere 17 (32-130017, hereafter marked as CEN17); and (iv) the SNRPN probe (32-190004). The TP53 probe identifies an archetypical tumor suppressor gene mapped to 17p13.1. The AML1 probe maps to chromosome 21q22, it is also used for the enumeration of chromosome 21. The centromere-specific probe CEN17, usually used for the enumeration of chromosome 17, identifies a specific non-coding repetitive pericentromeric array on this chromosome. The SNRPN probe identifies the Prader-Willi/Angelman syndrome-imprinted region on 15q11-13.
In-Situ Hybridization
Probes were diluted in Ingen's DenHyb solutions (Insitus Biotechnologies), in catalog #D001 (1:200) for CEN17 and in catalog #D003 (1:100) for AML1 and (1:50) for TP53 and SNRPN. We used these to replace the hybridization solutions supplied with the probes. Five μl of the probe solution were placed on the target area of the sample slides and covered with a 12 mm round silanized coverslip (Insitus Biotechnologies) and sealed with rubber cement. The slides were placed on a preheated aluminum slide tray (Insitus Biotechnologies) at 76°C and denatured for 6 min at that temperature. The slide-filled aluminum slide tray was then transferred into a HybBox (Insitus Biotechnologies) covered and allowed to hybridize overnight.
Detection
Post hybridization wash for probe TP53 was carried out by immersing the slides in 4 × saline sodium citrate (1× SSC = 150 mM NaCl, 15 mM sodium citrate) for five min at room temperature. Post hybridization washes for probe AML1 consisted of immersing the slides for 20 sec in 0.4 × SSC, pH 7.0 with 0.3% NP40 Nonidet P4 detergent), followed by 20 sec in 2.0 × SSC with 0.1% NP40 at 60°C in a shaking water bath. The post hybridization washing of the CEN17 was carried out in the same solutions as the AML1 probe, with the first wash carried out at 75°C for 2 min and the second at the same temperature for 1 min. After draining off excess liquid and brief drying, the slides were treated with 15 μl/slide of a solution of antifade containing 3 μg/ml of 4,6-diamidino-2-phenylindole (DAPI) as the counterstain (Vector Laboratories, Inc., Burlingame, CA, USA). Slides were covered with glass-coverslips (22 × 60 mm) and stored at -20°C till analyzed (which could take place anywhere between 1 hr and two days).
Cytogenetic Evaluation
Slides were analyzed blindly on an Olympus BH2 fluorescent microscope, using a triple band-pass filter (Chroma Technology, Brattleboro, VT, USA). The FISH replication assay was used here to follow the degree of synchrony in allelic replication (see Introduction). Accordingly, following hybridization, the fluorescence signals are divided into two categories, a single dot-like signal (designated: "singlet" or "S"), representing a yet non-replicated DNA sequence and a duplicated bipartite signal (designated: "doublet" or "D"), indicating a replicated sequence. Thus, two allelic counterparts replicating synchronously display a high frequency of cells with the two counterparts at the same replication status, either both non-replicated (SS cells; Figure 1A) or both replicated (DD cells; Figure 1B), and a low frequency of cells having one replicated and one non-replicated allele (SD cells; Figure 1C). In contrast, allelic counterparts replicating asynchronously exhibit a high frequency of SD cells. Hence, the SD cell frequency reveals the degree of asynchrony in allelic replication.
For the replication analyses, at least 100 interphase cells from each sample exhibiting two distinct well-defined fluorescence signals were scored for each treatment and probe under study. In each cell population, we noted the frequency (as a percentage, %) of SD cells out of the total population of interphase cells exhibiting two well defined fluorescence signals. We differentiated between a doublet signal and two singlets by ensuring that the two spots were not separated from each other by a distance greater than the diameter of the largest spot.
For detecting the level of aneuploidy, at least 200 interphase cells from each sample of blood preparations used for the replication analyses and treatments were screened for the numbers of chromosomes 17 and 21. For these determinations, we utilized, respectively, one-color FISH with the CEN17 and the AML1 probes. In each cell, the number of hybridization signals was recorded, and the frequency of cells with one signal was used to estimate the level of monosomic cells, the frequency of cells with three or more signals revealed the level of multisomy. Thus, the sum of the multisomy and the monosomy frequencies reflects the level of aneuploidy for the chromosome in question. The FISH interphase assay, as applied here, is currently the method of choice for detecting chromosome aneuploidy [reviewed in [21]].
Statistical Analysis
The statistical significance of the difference between two cell populations was determined using the two-tailed Student's t-test (Microsoft Excel).
Ethical Basis
This study was approved by the Institutional Helsinki Committee and the Israel Ministry of Health.