Laboratory characteristics of patients with variant Phiadelphia chromosome positive leukemia

Background Variant Philadelphia chromosomes are characterized by the involvement of another chromosome in addition to chromosome 9 or 22. To detect the difference between variant Philadelphia chromosomes positive leukemia and classic Philadelphia chromosomes positive leukemia. And to help diagnose and treat variant Philadelphia chromosomes positive leukemia. Methods In this study, Peripheral blood and bone morrow cell morphology test was used to analysis bone morphology of variant Ph positive patients. Karyotype analysis was used to find out variant Ph chromosomes. Flow cytometry analysis was used for immunology analysis. BCR/ABL was detected by PCR to monitor change of molecular genetics in variant Ph positive patients. Results From 48 patients with Ph positive leukemia, we found out 3 variant Ph positive leukemia. Compared with the classic Ph positive leukemia patients, the hemogram in the variant Ph positive leukemia patients was more variant for presenting hypomyelodysplasia or hyperactive. Compared with classic chronic myeloid leukemia which neutrophilic myelocyte, metamyelocyte and stab granulocyte is increasing in, both the morphological testing of bone marrow cells smear and flow cytometry analysis were indicated that proportion of myeloblast and promyelocyte increased in variant Philadelphia chromosomes leukemia. What’s more, the variant Ph with breakpoint 4q31 was the first report in leukemia patient. Same as the classic Ph positive leukemia patient, formal and effective treatment could prolong the survival of patients with variant Ph positive leukemia. Conclusions Compared with classic Ph positive leukemia, the hemogram was more variant in variant Ph. The myeloid morphology in the patients with variant Ph was more immature than that of in the patients with classic Ph. Reporting new cases of complex variant translocations, which can refer to new breakpoints that can eventually be recurrent and important for the understanding of this leukemia. Formal and effective treatment are necessary for variant Ph positve leukemia.

and mixed phenotype acute leukemia [1,2] . The Phildelphia chromosome is involved in the break-point cluster region-Abelson tyrosine kinase fusion gene, which encodes a constitutively active tyrosine kinase protein [3] . The Philadelphia chromosome is detected by karyotyping in around 90% of chronic myeloid leukemia (CML) patients, however, 5-10% may have variant types [4] . Variant Philadelphia chromosomes (Variant Ph) are characterized by the involvement of another chromosome in addition to chromosome 9 or 22. It can be a simple type of variant when one other chromosome is involved, or complex, in which two or more chromosomes take part in the translocation. Variant translocation could be formatted via either the one step or two step mechanisms [5] .
Most cases of CML are diagnosed in the chronic phase. The classic CML is Ph positive CML. In Ph positive CML, the leukocyte in peripheral blood was increasing obvisously and continuously. In patients with chronic myelogenous leukemia, the bone marrow is active or hyperactive, with an increased proportion of G/E(granulocyte/erythrocyte) [3] . The myelocytes proliferated as neutrophilic myelocyte, metamyelocyte and stab granulocyte. Erythrocytes proliferate at early stage and are inhibited at late stage. The thickness of granulocytes in bone marrow biopsy sections is usually 5-10 layers, compared with 2-3 layers in normal cases. When the acute myeloid line was changed, the protocells expressed the differentiation antigens associated with granular monocyte line, mononuclear line, megakaryocyte line and red line. The location of the BCR gene break point can affect the phenotype of the disease.
Imatinib should be considered in the same way as it is used for all patients with Ph-positive CML.
Nalatinib and dasatinib can prevent imatinib resistance due to tyrosine kinase mutations in most but not all Phiadelphia chromosome positive leukemia [5] . Few studies have reported the variant Ph in acute myeloid leukemia (AML). The BCR/ABL1 fusion protein (p210) was observed in 95% of Ph positive CML, whereas the p190 was appear in Ph positive ALL [6][7][8] .
The mechanism of variant Ph generation and the molecular bases of biological differences between classic Ph and variant Ph chromosomes are not fully understood [9] . Few comprehensive studies have reported the clinical characteristics of variant Philadelphia chromosomes or the molecular cytogenetic characterization involved among leukemia patients. Although they both have variant Phiadelphia translocation, the laboratory diagnostic characterization and prognosis are some different [10] . To find out the difference between classic Ph and variant Ph chromosomes leukemia and report new cases of complex variant translocations which may be involved new breakpoints, can eventually be help for diagnosing and treating these leukemia. morphology and proportions were analyzed [3,11] .

Karyotype analysis
Conventional cytogenetic analysis was performed on G-banded metaphase cells after having cultured bone marrow 24 hours using standard techniques. At least 20 metaphases with good quality banding were evaluated for each case when satisfactory cell cultures were available. A 320-band ideogram was considered as the standard level of resolution for the purpose of the present study. The karyotype was documented according to the International System for Human Cytogenetic Nomenclatures (ISCN 2013) [7] Flow cytometry analysis PCR for BCR/ABL BCR-ABL t(9;22) quantitative assay was performed in Sichuan kingmed center laboratory, Sichuan, China. Briefly, patient medulla RNA was isolated and reverse transcribed to complementary DNA (cDNA). The BCR/ABL1 and ABL1 reference gene sequences were amplified in duplicate using multiplexed quantitative real-time PCR. This assay can detect the major, minor, and micro BCR/ABL breakpoints and has an analytical sensitivity of better than 0.002%. [5] Results

Characteristics of variant Ph positive leukemia patients are some different
Three cases (6.38%) with variant t (9;22) were found out from 47 Ph positive CML & AML (Table 1).

Morphological tests of bone marrow cells smear
Compared with normal bone marrow image, bone marrow images with Ph+ leukemia patients had some characteristics: one CML case were hypomyelodysplasia like aplastic anemia (AA), and another CML case and one AML case is hyperactive, which is consistent with CBC of the three case (Fig1).

Discussion
In about 5-10% of CML cases, a complex translocation leaded to the formation of a variant Ph chromosome [12,13] . Three cases (6.38%) with variant t (9;22) were found out from 47 Ph positive CML & AML (Table 1) 1p36, 3p21, 5q31, 6p21, 9q22, 10q22, 11q13, 12p13, 17p13, 17q21, 17q25, 19q13, 21q22, 22q12 and 22q13 [14] . So 4q31 is first report up to now. Reporting new cases of complex variant translocations, which can refer to new breakpoints can eventually be recurrent and important for the understanding of this leukemia. What's more, studing new variant translocations and cryptic translocation can help for illuminating the differences and similarities between classic Ph positive leukemia and variant Ph positive leukemia, especially analyzing the diversity. Variant translocation could be via either the one-step or two-step mechanisms. some researchers [15][16][17][18] have introduced a one-step mechanism, wherein chromosome breakpoints occurs on three different chromosomes simultaneously in a three-, four-, or five-way translocation, then reciprocally rejoin at the same time.
Others [19,20] have confirmed a two-step mechanism, in which a standard two-way t(9;22) was followed by subsequent translocation involving additional chromosomes, and some recent studies [21] have reported both mechanisms in the same patient. We should use fluorescence in situ hybridization to judge the three patients' breakages in further study.
The karyotype and the combination of different FISH probes are essential to characterize complex variant Ph translocations [21][22] . In our article, conventional and molecular cytogenetic studies have allowed the characterize three complex variant Ph translocations. And the chromosomes most mainly involved were chromosomes 4 and 5. 85% of variant 9, 22 translocations located in the G-light bands (CG-richest areas) [22] . The CG richness areas reflected increasing in the density of the CpG islands, genes, repetitive elements, and recombination [23] .
In our study, one case was  [19,[23][24] .The available results provide evidence that the variant translocation in CML patients may affect response to imatinib therapy and patient prognosis [25,26] .So patients with a complex karyotype should be closely monitored for both side effects and disease progression under tyrosine kinase inhibitor treatment [28] .

Ethics approval and consent to participate
The authors have no ethical conflicts to disclose, and Ethics committee of affiliated hospital of North Sichuan Medical University approved this study.

Consent for publication
We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled.

Availability of data and material
The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors of this paper have no conflict of interests, including specific financial interests, relationships, and affiliations relevant to the subject matter or materials included.      PCR for BCR/ABL1 P210-type BCR-ABL fusion transcript appeared in bone morrow with patients2 and patients3.