It is important to detect PML/RARα rearrangement in patient with morphologic acute promyelocytic leukemia (APL). Rare cases of APL lacking the classic t(15;17) on routine cytogenetic studies have been described either as cases having complex variant translocations involving both chromosomes 15 and 17 with additional chromosome abnormalities and expressing PML/RARα transcript, or cases where neither chromosome 15 nor chromosome 17 is apparently involved, but with submicroscopic insertion leading to the expression of the PML/RARα transcript; these latter cases are considered as cryptic t(15;17).1 The analysis of these variant translocations is of great interest because they may be masking a cryptic t(15;17) and leading a true APL to being misdiagnosed as acute myeloid leukemia.2 To characterize the cytogenetic and molecular events in APL with cryptic t(15;17), we studied 160 consecutive newly diagnosed APL patients who were admitted in the hematology department of St Mary's Hospital from 2001 to 2006. The diagnosis of APL was made according to the World Health Organization classification system.1 Morphologic review confirmed the diagnosis of APL as described by Sainty et al.3 The immunophenotypic features including human leukocyte antigen-DR, CD34, CD13 and CD33 (Becton Dickinson, San Jose, CA, USA) were review. We performed chromosome analysis and fluorescence in situ hybridization (FISH) of the bone marrow cells at diagnosis. The probes used for FISH included (1) LSI PML/RARA dual-color, dual-fusion translocation probe (Vysis Inc., Des Plaines, IL, USA), which contained an ∼500 kb unique sequence probe for PML and an ∼700 kb probe for RARα; (2) PML/RARA t(15;17) dual-color, dual-fusion DNA probe (Qbiogene, Morgan Irvine, CA, USA), which contained an ∼600 kb probe for PML and an ∼450 kb probe for RARα; these probes can detect PML/RARα and RARα/PML fusion genes. (3) Iso 17q (MPO/p53) probe (Qbiogene) was used to identify the loss of p53 gene and the presence of two MPO gene signals (17q23.1) on isochromosome 17. A reverse transcriptase (RT)-PCR was also performed to survey whether PML/RARα had occurred or not. The sequences of the oligonucleotides used for RT-PCR are given for BCR1 of PML: 5′-IndexTermGTCTTCCTGCCCAACAGCAACC-3′; BCR3: 5′-IndexTermAGCTCTTGCATCACCCAGGGGA-3′; and RARα: 5′-IndexTermCTCACAGGCGCTGACCCCATAGT-3′. The amplified product was tested by gel electrophoresis and revealed a fragment of 186 bp for isoform BCR1 and 164 bp for BCR3.
Of the 160 patients, 143 (89.4%) revealed the presence of classic t(15;17) using conventional cytogenetic methods and FISH (Table 1). Sixteen patients (10.0%) did not have t(15;17) but revealed the underlying PML/RARα rearrangement, including the complex translocation of t(15;V;17) (25%) and cryptic t(15;17) (75%). The ratio of complex translocation and cryptic t(15;17) was 33.3–66.7% in the previous report.2 Clinical and biological information of these patients with cryptic t(15;17) is summarized in Table 2. The metaphase FISH and molecular findings were consistent with PML/RARα rearrangements being mediated by insertion events. In 4 of 12 patients (33.3%), a fusion or colocalization signal reflecting the formation of PML/RARα was localized to 15q (cases 1–4). In 6 of 12 patients (50%), formation of the PML/RARα fusion gene was localized to 17q (case 5–9). In the two remaining patients (16.7%), the FISH study failed to detect PML/RARα fusion; however, RT-PCR results proved the presence of PML/RARα fusion gene. They were considered as probable insertion. These results are different from Grimwade et al.2 (53.6, 17.9 and 28.6%). Patients with cryptic t(15;17) was not different in immunophenotype compared with the classic t(15;17) including expression of CD34 (data not shown) and showed good response to all-trans-retinoic acid (ATRA) therapy as previous reports.2, 3 Clinical follow-up was available for 11 patients. All patients except one were treated with combination chemotherapy in addition to ATRA and achieved complete remission. Case 11 was initially misdiagnosed as acute myeloid leukemia with differentiation because karyotype and FISH were normal, whereas the morphology was consistent with hypogranular APL. Initial RT-PCR was omitted. Three patients died; two died due to transplantation complication and one died in relapse state. Initial diagnosis is important because initiation of therapy is highly dependent on morphology and available diagnostic tools. However, conventional karyotype never detected cryptic t(15;17). Some large FISH probes did not detect PML/RARα fusion gene due to hiding the small insertional signal under bright fluorescence. Intensive reading was required to detect cryptic insertion by FISH. It can be occurred in RT-PCR to fail to detect PML/RARα transcript. The lack of fusion transcripts using RT-PCR can be explained either with the combination of variable positions of the PML breakpoints and insufficient variability of primers, or with the insertion of PML material near to RARα, and not within it.4 Therefore, morphologic, cytogenetic and molecular analyses such as FISH and RT-PCR should be combined to diagnose APL accurately.
Interestingly, we found three patients with new recurrent additional cytogenetic abnormality as i(17q) containing cryptic t(15;17). Among them, two patients revealed PML/RARα fusion on 17q; hence, two fusion signals were found on i(17q). One patient had one PML/RARα fusion on 15q. The presence of two MPO signals on i(17)(q10) made it possible to differentiate from ider(17)t(15;17). Only one APL case having the karyotype 46,XX,i(17)(q10) with the PML/RARα fusion gene on both arms of i(17) was described.5 Two APL cases were included in the review of an previous article about i(17)(q10), but they did not confirm the presence of PML/RARα.6 We cannot deny the possibility of i(17)(q10) with cryptic t(15;17) in those cases. The previous multicenter study did not report the incidence of i(17)(q10) with cryptic t(15;17).2 In APL with classic t(15;17), ider(17)t(15;17) is common additional chromosomal abnormality.7 We set up a hypothesis stating that the comparison of i(17)(q10) to cryptic t(15;17) should be considered as a counterpart of ider(17) to t(15;17) and can be secondary event after submicroscopic insertion. The ratio of i(17)(q10) to cryptic t(15;17) was much higher than that of ider(17) to classic t(15;17) (25 vs 4.9%). Our report can call an attention to detect this hidden recurrent additional abnormality. A continued progression toward a more careful approach in addition to a workup will be helpful to clarify the cytogenetic and molecular event of APL.
Jaffe E, Harris N, Stein H, Wardiman J . World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. IARC Press: Lyon, 2001, pp 84–86.
Grimwade D, Biondi A, Mozziconacci M-J, Hagemeijer A, Berger R, Neat M et al. Characterization of acute promyelocytic leukemia cases lacking the classic t(15;17): results of the European Working Party. Blood 2000; 96: 1297–1308.
Sainty D, Liso V, Cantù-Rajnoldi A, Head D, Mozziconacci M-J, Arnoulet C et al. A new morphological classification system for acute promyelocytic leukemia distinguishes cases with underlying PLZF/RARA rearrangements. Blood 2000; 96: 1287–1296.
Tchinda J, Volpert S, Liersch R, Zühlsdorf M, Serve H, Neumann T et al. A cryptic insertion (17;15) on both chromosomes 17 with lack of PML-RARA expression in a case of atypical acute promyelocytic leukemia. Leukemia 2004; 18: 183–186.
Lee G, Christina S, Tien S, Ghafar A, Hwang W, Lim L et al. Acute promyelocytic leukemia with PML–RARA fusion on i(17q) and therapy-related acute myeloid leukemia. Cancer Genet Cytogenet 2005; 159: 129–136.
Becher R, Carbonell F, Bartram C . Isochromosome 17q in Ph1-negative leukemia: a clinical, cytogenetic, and molecular study. Blood 1990; 75: 1679–1683.
de Botton S, Chevret S, Sanz M, Dombret H, Thomas X, Guerci A et al. Additional chromosomal abnormalities in patients with acute promyelocytic leukaemia (APL) do not confer poor prognosis: results of APL 93 trial. Br J Haematol 2000; 111: 801–806.
We acknowledge the financial support of the Catholic Medical Center Research Foundation.
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