|Year : 2015 | Volume
| Issue : 1 | Page : 60-63
Fluroscence in-situ hybridization negative PML/RARA: A cryptic puzzle
Manasi Chetan Mundada1, Faiq Ahmed1, G Sandhya Devi1, Sudha Murthy2, M. V. T. Mohan2
1 Department of Lab Medicine, Basavatarakam Indo-American Cancer Hospital and Research Institute, Hyderabad, Telangana, India
2 Department of Medical Oncology, Basavatarakam Indo-American Cancer Hospital and Research Institute, Hyderabad, Telangana, India
|Date of Web Publication||5-Jun-2015|
Dr. Manasi Chetan Mundada
Basavatarakam Indo - American Cancer Hospital and Research Institute, Banjara Hills, Hyderabad, Telangana- 500 034
Source of Support: None, Conflict of Interest: None
Acute promyelocytic leukemia (APL) has defined biology and clinical course that is, distinct from the other forms of acute myelogenous leukemia. It may present with potentially devastating coagulopathy and the sensitivity to retinoid differentiating agents, including all-trans retinoic acid and arsenic trioxide, hence a fast and definite diagnosis is imperative. Reciprocal 15, 17 translocation creates a PML/RARA fusion gene on the derivative chromosome 15, which can be detected by various molecular tests such as cytogenetics, fluroscence in-situ hybridization (FISH), reverse transcriptase-polymerase chain reaction. We present here a diagnostically challenging case, both morphologically and immunophenotypically proven to be APL, which was negative for the PML/RARA by FISH.
Keywords: Acute promyelocytic leukemia, fluorescence in-situ hybridization, PML/RARA
|How to cite this article:|
Mundada MC, Ahmed F, Devi G S, Murthy S, Mohan M. Fluroscence in-situ hybridization negative PML/RARA: A cryptic puzzle. Indian J Health Sci Biomed Res 2015;8:60-3
|How to cite this URL:|
Mundada MC, Ahmed F, Devi G S, Murthy S, Mohan M. Fluroscence in-situ hybridization negative PML/RARA: A cryptic puzzle. Indian J Health Sci Biomed Res [serial online] 2015 [cited 2020 Jan 22];8:60-3. Available from: http://www.ijournalhs.org/text.asp?2015/8/1/60/158238
| Introduction|| |
Acute promyelocytic leukemia (APL) has a defined biology and clinical course, that is, distinct from other forms of acute myelogenous leukemia (AML), may present with potentially devastating coagulopathy and sensitivity to retinoid differentiating agents including all-trans retinoic acid (ATRA) and arsenic trioxide. Hence, a fast and definite diagnosis is imperative. APL is a rare subtype of acute myeloid leukemia, characterized by the presence of the reciprocal 15; 17 translocation involving the PML gene on 15q24, and RARA gene on 17q21 in more than 90% of cases. This translocation creates a PML/RARA fusion gene on the derivative chromosome 15, which can be detected by various molecular tests such as cytogenetics, fluorescence in-situ hybridization (FISH), reverse transcriptase-polymerase chain reaction (RT-PCR). However, cases showing complex PML/RARA translocations or variant translocation have been described. Here a diagnostically challenging, morphologically, and immunophenotypically proven APL case negative for the PML/RARA rearrangement by FISH is presented.
| Case Report|| |
A 30-year-old male was referred to the Medical Oncology Department for evaluation of fever associated with bleeding gums and easy bruisability.
Hemogram showed hemoglobin of 61 g/L, total leukocyte count (TLC) was 4.7 × 10 9 /mm 3 and platelets as 8 × 10 9 /mm 3 . Peripheral blood examination revealed the red blood cells to be normocytic normochromic. White blood cells showed a normal count with a predominant population of the large cells, 2½-3 times the size of mature lymphocytes with the moderate nucleo-cytoplasmic ratio. The nuclear contours were regular, and at places bilobed nucleus was evident. Cytoplasm showed dense basophilic granules, one to multiple Auer rods More Details. Platelets were severely reduced [Figure 1]a. Cytochemical staining showed strong positivity for myeloperoxidase [Figure 1]b while periodic acid schiff was negative. Immunophenotyping performed on four color multiparameter flow cytometer showed a high forward and side scatter. The neoplastic cells showed moderate positivity for CD45, CD13 and CD33, and negative for CD34, CD117, HLA-DR, CD14, CD56, pan B, and T cell markers [Figure 2]. The diagnosis of APL was rendered and for confirmation of PML/RARA rearrangement FISH was suggested. Interphase FISH for PML/RARA rearrangement was performed on bone marrow using Vysis LSI dual color dual fusion translocation probe. The cells were surprisingly negative for the characteristic PML/RARA rearrangement [Figure 3].
|Figure 1: (a) Leishman stained peripheral blood smears showing hypergranular forms of promyelocytes (×400) (b) Cytochemical stain myeloperoxidase highlights the granules in promyelocytes (×400)|
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|Figure 2: Immunophenotyping scatter plots showing the high side scatter with positive antigen markers for myeloid lineage antigens|
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|Figure 3: Fluorescent in-situ hybridization by Vysis probe showing a negative/normal signal pattern with two red and two green signals|
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Further confirmation for PML/RARA transcript by qualitative PCR, which was outsourced, showed positivity for the PML/RARA transcript-bcr1.
Biochemical investigations at presentation revealed, activated partial thromboplastin (APPT) time and PT values to be 39 (control 30) and 18.9 (control 15), respectively. Fibrinogen levels were 268 mg/dL (range: 150-400). Liver function tests and serum creatinine were within the normal limits.
Patient was started on supportive measures and ATRA at a dose of 45 mg/m 2 /day with which the TLC started increasing, clinically suggestive of the release of maturation arrest with ATRA. Chemotherapy comprised of daunorubicin 110 mg/square body surface area and dexamethasone 12 mg/kg body weight. Supportive care as antibiotics, fresh frozen plasma, platelets transfusion, and packed cells were transfused.
| Discussion|| |
Acute promyelocytic leukemia constitutes 5-8% of AML and is characterized by proliferation of atypical promyelocytes in the bone marrow and peripheral blood.  APL is diagnosed based on the morphology, cytochemical stains, immunophenotyping, and confirmed by the presence of t (15;17) by conventional cytogenetics or PML/RARA rearrangement by FISH/RT-PCR. The present case was diagnosed as APL based on the morphology, supportive cytochemistry, and immunophenotypic findings. However, FISH was negative for PML/RARA rearrangement. Qualitative RT-PCR confirmed the presence of PML/RARA transcript of bcr1. Among the number of APL cases diagnosed at this center this is first case showing discordance between FISH result and the morphology.
In an attempt to understand the reasons for this discrepancy literature search was performed. Grimwade and Lo Coco indicated that 92% of APL cases demonstrated PML/RARA rearrangement. Other aberrant genetic abnormalities include simple or more complex variants, such as insertions of RARA into PML or vice versa (2%), RARA fused to partner genes other than PML (4%) or in 1% cases with no RARA rearrangement. 
Campbell et al. described 10 cases of APL without a t (15;17) that appear to have produced a PML/RARA fusion gene by insertion of a small segment of RARA into the PML gene of cytogenetically normal chromosome 15. The incidence of cryptic insertions in APL was described to be 5%.  The authors were unable to demonstrate the PML/RARA rearrangement with Abbott LSI PML/RARA dual color dual fusion translocation probe despite RT-PCR showing the presence of PML/RARA transcript. The same rearrangement was confirmed by the Cytocell probe. Further, the description of Abbott and Cytocell probes was checked. The evaluation revealed Abbott is a large probe and the discrepancy between the size of the Vysis PML signal and very small RARA segment inserted into 15q24 allowed the PML signal intensity to quench the RARA signal.  In the present study, Vysis PML/RARA probe, like Abbott is a large probe. LSI PML probe for chromosome 15 is 180 kb and 335 kb on either side of the PML loci including all of the known BCR regions of PML, labeled in spectrum orange. RARA labeled in spectrum green is approximately 700 kb of chromosome 17 spanning all the breakpoint regions. A positive case would show one green, one red, and two fusion (PML/RARA and RARA/PML) signal pattern. With Cytocell PML/RARA translocation dual fusion probe, the extra signal probe is smaller, of 40 kb in size. An abnormal cell revealed two green signals plus a tiny third green signal representing an insertion. In the current case using the Vysis probe no extra signal could be visualized. Kim et al. suggested, in large probes the PML/RARA fusion signals are not visible due to hiding of the small insertions behind bright fluorescence  and hence the less disparate intensities of the two signals using the Cytocell probes allowed the insertion to be visualized. Previous studies also demonstrated the shortcoming of the Vysis probe wherein small insertion at 3' end of PML went unnoticed due to under representation of this area on Vysis probe.  Welch et al. in 2011 described cases wherein FISH for APL associated gene rearrangements was negative. These cases were attributed to masked or cryptic translocations occurring at the submicroscopic level. 
Koshy et al. described 1 case wherein the FISH failed to detect the translocation however was confirmed by RT-PCR. The rare cryptic fusions often associated with small genomic insertions are best detected by RT polymerase and microarray that enable the detection of the amount of material inserted. The cryptic PML/RARA fusion may be due to complex translocations involving chromosomes 15 or 17 and submicroscopic insertion of RARA in PML gene or vice versa the detection of which is beyond the resolution signal leading to false negative results. 
This patient was started on ATRA along with other supportive measures such as blood products and platelet transfusions. Daunorubicin was added later, his headache worsened over time and a magnetic resonance imaging of the brain revealed a small infarct in the right occipital region. He was managed with controlled anticoagulation, but he succumbed to worsening infarction with mass effect.
Take home message
Accurate and timely diagnosis of APL is critical in patient management. FISH for PML/RARA rearrangement plays a vital role in confirming the diagnosis. The present case and evidence from the literature indicate that diagnosis of cases with cryptic PML/RARA is challenging. In the diagnostic setup, careful examination of the interphase signal pattern is necessary, and use of other molecular diagnostic modalities RT-PCR or availability of alternate probes is imperative in morphologically positive, and in rare instances PML/RARA FISH negative cases.
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[Figure 1], [Figure 2], [Figure 3]