Nikhil Rabade1#, Goutham Raval1#, Shruti Chaudhary1#, PG Subramanian1, Rohan Kodgule1, Swapnali Joshi1, Prashant Tembhare1, Syed K. Hasan1, Hasmukh Jain2, Manju Sengar2, Gaurav Narula2, Shripad Banavali2, Pratibha Amare Kadam3, Dhanalaxmi Shetty3, Sumeet Gujral1 and Nikhil Patkar1.
1 Hematopathology laboratory, Department of Pathology, Tata Memorial Centre, Mumbai.
2 Department of Medical Oncology, Tata Memorial Centre, Mumbai.
3 Department of Cancer Cytogenetics, Tata Memorial Centre, Mumbai
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breakpoints and variant APL cases involving alternative chromosomal
aberrations are seen in a small subset of acute promyelocytic leukemia
(APL) patients. Over seven different partner genes for RARA have been
described. Although rare, these variants prove to be a diagnostic
challenge and require a combination of advanced cytogenetic and
molecular techniques for accurate characterization. Heterogeneity
occurs not only at the molecular level but also at clinico-pathological
level influencing treatment response and outcome. In this case series,
we describe the molecular heterogeneity of APL with a focus on seven
variant APL cases from a single tertiary cancer center in India over a
period of two and a half years.
Materials and Methods
|Figure 1. Distribution of APL cases as per the fusion transcript type.|
|Table 1. Clinico-pathological features of APL cases.|
|Figure 2. A&B – Case 1. Abnormal promyelocytes with rounded nuclei and dense cytoplasmic granularity and hypolobated neutrophils. C&D – Case 2. Relatively scanty cytoplasmic granularity of the promyelocytes. E&F – Case 5. Hypolobulated and hypogranular myeloid cells along with abnormal promyelocytes.
|Figure 3. Cellular Morphology and PML-RARA fusion transcript detection. Abnormal promyelocytes with characteristic bilobed nuclei (B) and presence of abundant cytoplasmic granules which showed strong cytochemical myeloperoxidase positivity (D) is seen in the uppermost panel. Some unusual myeloid differentiation is also seen (A & C). PCR amplicon size (480 bp) of novel Bcr3 PML-RARA transcript on agarose gel (middle panel) and capillary (lower panel) electrophoresis.|
|Figure 4. Characterization of novel Bcr 3 PML-RARA variant by Sanger sequencing (Case 6). Schematic representation of novel PML-RARA variant transcript is shown in the upper panel. PML gene exons are shown the squares and RARA gene exons are shown in the circles. Insertion of 40 nucleotides from RARA intron 2 is shown as a shaded black circle. Exact patient sequences are shown in dashed box below. The cDNA and gDNA (lower panels) from the patient were sequenced by Sanger sequencing and aligned to the exonic and/or intronic sequences of PML and RARA genes.|
|Figure 5. Characterization of Bcr 2 PML-RARA variant (Case 7). Agarose gel electrophoresis (upper panel) of our case (Case 7) along with known positive control (PC), biologically negative control (BNC) and a non-template control (NTC). Sanger sequencing (lower panel) of the same case shows fusion of PML exon 6 with RARA exon 3 and addition of 126 nucleotides from RARA intron 2.|