1Clinical Department of Laboratory Diagnostics, Division for Cytogenetics, University Hospital Centre Zagreb, Zagreb, Croatia.
2Department of Laboratory Diagnostics, General Hospital “Dr. Josip Benčević”, Slavonski Brod, Croatia.
3Clinical Department of Laboratory Diagnostics, Division of Laboratory Hematology and Coagulation, University Hospital Centre Zagreb, Zagreb, Croatia.
4Department of Medical Biochemistry and Hematology, University of Zagreb Faculty of Pharmacy and Biochemistry, Zagreb, Croatia.
To analyze the frequency of atypical fluorescence
The study included bone marrow and peripheral blood samples from 122 patients with newly diagnosed chronic myeloid leukemia. Detection of the
Variant translocation was determined in 10 samples and a deletion on the derivative chromosome 9 (del/der(9)) was found in 20 samples. The rates of CCyR and MMR were similar between patients with reciprocal translocation, variant translocation, deletion of derivative
The frequencies of variant translocation and del/der(9) in the present study agree with the results of other studies performed worldwide. No differences were observed in the rates of CCyR and MMR between patients with atypical patterns and reciprocal translocation.
Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by a clonal disorder of hematopoietic stem cells. CML appears in 15–20% of all cases of leukemia, but it is very rare in children and adolescents, where it accounts for less than 2% of cases .
CML is genetically characterized by a reciprocal translocation that results in an exchange of genetic material between the long arm of chromosome 22 (22q−) and the long arm of chromosome 9 (9q+). This exchange is referred to as translocation t(9;22)(q34;q11.2) and creates a derivative chromosome 22 that is known as the Philadelphia chromosome (Ph). At the molecular level, this reciprocal exchange involves the Abelson oncogene (
The standard treatment options for patients in the chronic phase of CML were previously hydroxyurea, interferon-α, or allogenic stem cell transplantation . The treatment of CML was revolutionized in 2001 by the introduction of imatinib mesylate, which is a
The Ph chromosome is detected in 90% of patients in the chronic phase of CML . In the clinical diagnosis of CML, the ‘state of the art’ methods comprise classical banding techniques, fluorescence
The aim of this study was to analyze the frequencies of variant translocation and deletion on derivative chromosome 9 (del/der(9)) in newly diagnosed CML patients in a Croatian population. The complete cytogenetic response (CCyR) and the major molecular response (MMR) were also estimated at 12 months after the onset of TKI therapy.
A retrospective study was conducted from January 2013 to October 2016 (total duration 3 yr and 9 mo) at University Hospital Centre Zagreb. Samples from 122 newly diagnosed Ph-positive CML patients were included. These patients comprised 53 (43%) women and 69 (57%) men (
Conventional cytogenetics and FISH were performed at the Division for Cytogenetics, University Hospital Centre, Zagreb. Conventional cytogenetics was performed using the standard Giemsa banding method. Analysis was performed on bone marrow cells after short-term culture (24 h). The cells were treated with a colchicine and hypotonic solution then fixed and washed in methanol-acetic acid (3:1). After that, the cells were resuspended in fixative and dropped onto slides . At least 20 metaphases per sample were analyzed and captured using a fluorescence light microscope (Carl Zeiss Microlmaging GmbH, Gottingen, Germany), and image analysis was performed using the Ikaros software package (MetaSystems, Heidelberg, Germany). Karyotypes were described according to the criteria of the International System for Human Cytogenetic Nomenclature (ISCN 2009, ISCN 2013) .
The FISH procedure was performed on a fixed cell suspension obtained with a conventional cytogenetic method on microscope slides . A mixture of probe, buffer, and water was applied onto the fixed BM cells according to the manufacturer's instructions. The processes of denaturation (1 min at 73℃) and hybridization (24 h at 37℃) were performed using an automated machine (HYBrite, Abbott Laboratories, IL, USA). Non-specifically bound probe was washed in a water bath for 2 min with special detergent solutions. After the microscope slide was allowed to air dry, it was treated with the DNA-intercalating dye 4,6-diamidino-2-phenylindole (DAPI) and a glass cover slip was applied.
The Vysis LSI
A sample of 200 interphase cells for each patient was analyzed using a fluorescence light microscope (Carl Zeiss Microlmaging GmbH, Gottingen, Germany) under ×1,000 magnification with the use of an immersion oil and optical filters (DAPI, red, green, and triplet- a filter that can see DAPI, red, and green signals simultaneously). The images were processed using the ISIS computer program (ISIS software, Altlussheim, Germany). The results were expressed as the percentage of Ph-positive interphase cells per hundred counts.
The cutoff value was determined with the FISH method in 20 patients with a diagnosis unrelated to CML and it was 1%. If the proportion of
According to established recommendations, the BM samples for cytogenetic analysis and FISH were obtained at diagnosis, 3 months later, and then every 6 months until the achievement of CCyR .
After that, they were analyzed every 12 months. Despite the recommendation, the sample was sometimes sent only for RQ-PCR and therefore a smaller number of cases were monitored for CCyR than for MMR.
RQ-PCR was performed at the Laboratory for Molecular Hematology, University Hospital Centre, Zagreb standardized for reporting results according to the International Scale (IS). For each patient, duplicate samples of 2×107 PB leukocytes were homogenized with TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), followed by the extraction of RNA. The reverse transcription of 2 µg RNA was performed using the high-capacity cDNA reverse transcription kit with RNase inhibitor (Thermo Fisher Scientific). The amplifications of the fusion gene
Categorical data was compared using Fisher's exact test. Time to CCyR and time to MMR were calculated from the beginning of the treatment and the next 12 months using the Kaplan-Meier method and described with cumulative incidence curves.
Among 122 patients with positive BCR-ABL1 rearrangement, the median proportion of BCR-ABL1 copies in interphase nuclei was 85% (range, 2–97). Ninety-two (75%) patients showed reciprocal t(9;22) (q34;q11.2) and 30 (25%) showed atypical FISH signals.
All patients in this study started with imatinib mesylate therapy at the dose of 400 mg/day. If their response to therapy was not sufficient, they were switched to nilotinib or dasatinib.
In this study, 70 (57%) samples were evaluated using conventional cytogenetics. Four (40%) of ten cases of variant t(9;22) showed the Ph chromosome on karyogram analysis and six cases (60%) did not show the Ph chromosome due to a lack of cells undergoing cell division. Newly diagnosed CML patients typically have a high level of white blood cells, resulting in a dense BM sample and consequently a low quality of metaphases, which can impede the success of karyotyping.
The karyogram of case number (No.) 2 showed t(9;22) with chromosome 1 involved in the translocation, resulting in t(1;9;22)(p35;q34;q11.2). The karyogram of case no. 10 showed that the third chromosome involved in t(9;22) was chromosome 9, resulting in t(9;9;22)(q34;q34:q11.2) (Fig. 1). Two cases (No. 1 and No. 4) only showed t(9;22) and no involvement of the third chromosome was detected. The submicroscopic deletion of del/der(9) cannot be detected by conventional cytogenetic methods and karyotype analysis.
All the samples were successfully analyzed with FISH. Generally, in negative interphase nuclei for t(9;22), the signal pattern consisted of two red signals (two normal copies of chromosome 9) and two green signals (two normal copies of chromosome 22), which was termed 2R2G (Fig. 2A). With the dual-color dual-fusion BCR/ABL probe, the positive reciprocal t(9;22) signal pattern revealed two fusion signals (derivative chromosome 9 and derivative chromosome 22), one red signal (normal chromosome 9), and one green signal (normal chromosome 22), which was termed 2F1R1G (Fig. 2B). Cases with an atypical FISH signal pattern comprising one fusion signal (derivative chromosome 22), two red signals (normal and derivative chromosome 9), and two green signals (normal chromosome 22 and a third involved chromosome) were termed 1F2R2G (Fig. 2C) and represent variant t(9;22). We noted variant t(9;22) in 10 (8.2%) of the samples (5 females and 5 males). Their median age was 49 years (range, 29–69). The median proportion of
The next three atypical FISH signals described represented deletions. Ten (8.2%) samples of reciprocal t(9;22) with additional cytogenetic abnormalities such as an
Four (3.3%) samples with a
Of the 122 cases with a diagnosis of CML, 51 were included in our database for tracking the cytogenetic response. CCyR was identified in 0% of Ph-positive interphase nuclei on FISH . The results for the cytogenetic response are shown in Table 3 and Fig. 3 overall, 28 (54.9%) patients achieved CCyR within 12 months of starting the treatment, while 23 (45.1%) had not responded to therapy 12 months after starting the treatment.
For MMR, 76 of the 122 patients were monitored. MMR was defined as a
To our knowledge, this is the first study that evaluated the incidence and molecular-cytogenetic response to TKI of variant t(9;22) and del/der(9) in Croatian CML cases. In the present study, variant translocation occurred in 8.2% of patients, similar to results of Marzocchi et al. who found 5–10% of newly diagnosed CML cases with a variant translocation. The mechanisms of the variant translocations are not fully clear, but Marzocchi et al.  have suggested 1-step and 2-step mechanisms (Fig. 5).
Nevertheless, 83.3% patients with variant t(9;22) achieved CCyR and 62.5% achieved MMR. In our study, the presence of variant translocation did not have an influence on CCyR or MMR, which were achieved within a year. Compared to reciprocal t(9;22) patients, 50.0% of them reached CCyR and 64.8% reached MMR. For all patients with variant translocation, response durations were similar to those of patients with reciprocal Ph translocation.
Significance of del/der(9) in patients on TKI therapy is still uncertain, but a number of studies showed that this deletion occurs at the same time as the formation of Ph chromosome . Sinclair et al.  and Huntly et al.  suggested a significant association between the presence of der/del(9) and a poor prognosis, but yet only for patients who have received interferon. For this reason, the European LeukemiaNet recommendations considered der/del(9) as a candidate for adverse prognostic factor in 2006, which requires a careful monitoring of patients on TKI . Present study showed 16.4% of cases with del/der(9), similar to published results of 10–15% patients with CML .
In this study, data showed that 4.9% of patients had
Morel et al.  reported 9% and Lee et al.  demonstrated 7% patients with
The only difference in achievement of MMR was observed for deletion of
This study showed that the percentage of variant translocation and del/der(9) in Croatian population is in agreement with the data from previously published studies from around the world. A number of studies agree that atypical patterns have no impact on the prognosis of patients with CML . In our series of patients, it was not conclusive that a significant difference in achievement of CCyR and MMR exists between reciprocal translocation and variant translocation, deletion of
We would like to thank Ivana Franić Šimić and Margareta Radić Antolic from University Hospital Centre Zagreb for their assistance and support.