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
Received: August 24, 2017
Accepted: November 6, 2017
Mediterr J Hematol Infect Dis 2018, 10(1): e2017027 DOI 10.4084/MJHID.2018.002
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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.
Approximately 8% of APL cases lack the characteristic t(15;17)(q22;q12) translocation on cytogenetic testing. These include cases with cryptic translocations or those with variant RARA translocations. Variant translocations involve fusion of RARA with one of eleven partner genes which include PLZF or ZBTB16 (chromosome 11q23), NPM (5q35), NuMa (11q13), STAT5b (17q21.1-21.2), PRKAR1A (17q24), FIP1L1 (4q12) and BCOR (X). Recently OBFC2A, NABP1 and IRF2BP2 have also been reported to fuse with RARA. ZBTB16-RARA fusion is a rare entity seen in a minority of patients with APL. Unlike classical APL, variants involving ZBTB16 lack the differentiation response to ATRA therapy and carry an unfavorable prognosis.
Identification of the above mention fusion genes requires a combination of FISH, conventional karyotyping and molecular techniques, which are not readily available in most diagnostic laboratories in India. The scarcity of diagnostic centers possessing these advanced techniques, coupled with the rarity of this disease has resulted in lack of data describing such cases from India. Thus, we illustrate the molecular heterogeneity of APL from a tertiary cancer center in India over a period of two and a half years. We describe the clinicopathological features of variant APL cases along with two novel PML-RARA variants.
Materials and Methods
RNA extraction and cDNA synthesis. RNA was extracted from bone marrow samples after red blood cell (RBC) lysis using RNA blood mini kit (Qiagen, Germany). cDNA was obtained from RNA using the ‘High capacity’ reverse transcriptase cDNA synthesis kit (Applied Biosystems, CA, USA)
Fusion transcript detection and Real-time quantitative PCR (RT-qPCR). ZBTB16-RARA fusion transcript was identified using a previously described real-time quantitative PCR(RT-qPCR) assay on a Roche Light Cycler 96. The delta-delta Ct method was applied to determine the post-induction ZBTB16-RARA fusion transcript copy number. Each sample was run in triplicate along with ABL1 as the control gene.
The PML-RARA fusion transcript was identified by using the reverse transcriptase PCR (RT-PCR) BIOMED-1 protocol as described by van Dongen et al.. Plasmid standards for the novel PML-RARA variant, ranging from 106 to 101 were prepared in-house by cloning RT-PCR amplicons (see below). We followed Europe against cancer (EAC) guidelines for primer, probe design and RT-qPCR assay for quantifying PML-RARA fusion transcripts.
Sequencing and cloning. The PML-RARA variant RT-PCR products were purified with a SapExo solution (Life Technologies, CA, USA) and incubated at 37 degrees C for 15 min followed by 80C for 15 mins. Purified products were cloned into a pJET 1.2 vector by using the Clone JET PCR Cloning kit (Thermo Scientific, USA) to give a final volume of 20 ul of cloned product. TOP HAT DH5alpha competent cells (Invitrogen, CA, USA) were transformed with 10ul of cloned product and selected for recombinants by antibiotic selection. DNA from the resultant clones was extracted by using a QIAprep Spin Miniprep Kit (Qiagen, Germany) and quantified by NanoDrop 2000 spectrophotometer. The PCR product and the cloned product were sequenced by Sanger sequencing. The following sequences from Ensemble were used as reference: PML-001 (ENST00000268058.7) and RARA-007 (ENST00000425707.7).
Long-range genomic DNA PCR. Long-range PCR for the PML-RARA variant was performed by using TaKaRa LA Taq kit according to the manufacturer’s protocol. Briefly, 2 ul of genomic DNA (gDNA) was used as a template and subjected to PCR at 94C for 1 min, 98C for 10 sec & 68C for 15 mins (30 cycles) and 72C for 10 mins. One forward primer spanning PML exon 3 and eight reverse primers for RARA intron 2 were used for PCR. The PCR products were run on a 1% agarose gel and DNA from the required band was extracted by using QIAquick Gel Extraction kit (Qiagen, Germany). The extracted DNA was subjected to Sanger sequencing using the same primers.
|Figure 1. Distribution of APL cases as per the fusion transcript type.|
Case 1. A 15-year-old boy complained of abdominal pain, weakness and intermittent fever. Laboratory investigation values are depicted in Table 1. Bone marrow examination revealed abnormal promyelocytes with regular, round to oval nuclei, (lacking the classical bilobed appearance) dense cytoplasmic granularity, the absence of Auer rods but strong cytochemical myeloperoxidase (MPO) positivity (Figure 2, A & B). Few Pelger-like neutrophils were also observed along with maturing myeloid cells. The patient did not develop any bleeding manifestations. FCI revealed cells having high side scatter and dim CD45 expression, heterogeneous CD13, and homogeneous bright CD33, along with lack of CD34, HLA-DR, and CD56 expression. FISH was negative for t(15;17). The RARA break-apart probe detected a variant RARA translocation. PCR for PML-RARA fusion transcripts was negative as well. ZBTB16 as the partner gene for variant RARA was confirmed with PCR. The patient received induction with Idarubicin and arsenic trioxide (ATO). Forty-five days post induction remission was not achieved.
FISH showed variant RARA positivity in 52% cells. RT-qPCR showed mean threshold cycle (Ct) value of 26.78 against a baseline Ct value of 21.42 for ZBTB16-RARA and 21.03 for ABL1. The patient further received three cycles of high dose cytarabine (HiDAC) following which complete remission was achieved with no detectable ZBTB16-RARA fusion transcripts.
Case 2. A 38-year-old man with complaints of easy fatigability, dyspnea, and intermittent fever was referred with a suspicion of APL for molecular testing. FISH detected a variant RARA translocation which was confirmed to be ZBTB16-RARA by PCR. However, the morphological features differed from the previous case. The abnormal promyelocytes with regular nuclei showed the presence of Auer rods but had abundant cytoplasm with scanty granularity along with the presence of Pelger-like neutrophils (Figure 2, C & D).
The patient received ATO based induction therapy but responded poorly and failed to achieve remission. The post induction Ct value (25.02) by RT-qPCR was similar to the baseline one (23.65). However, the patient died shortly after completing two months of therapy.
Case 3. A 45-year-old man was referred for molecular testing to confirm the morphological suspicion of APL. Fever and easy fatigability were the only presenting symptoms. Morphological features were similar to case 1, as highlighted by the presence of abnormal promyelocytes containing round nuclei with regular nuclear contours, abundant granulated cytoplasm and a lack of Auer rods. In contrast to classical APL cases maturing myeloid cells were also present along with a few hypolobated or Pelger-like neutrophils. Dual fusion FISH for PML-RARA was negative, but the break-apart probe revealed RARA variant translocation. ZBTB16-RARA fusion was confirmed by PCR. RT-qPCR showed a baseline mean Ct value of 21.64. Although the patient was started on induction with ATO, he was lost to follow up subsequently.
Case 4. The patient is a 36-year-old gentleman with relapsed APL referred to our institute for further management. A detailed history revealed that the patient was first presented with fever and rash in February 2016 and morphological features on peripheral smear raised the suspicion of APL. However, PML-RARA was not detected by molecular testing (details of PML-RARA or variant RARA testing not available). The patient was started on induction therapy with daunorubicin and cytarabine followed by three cycles of HiDAC. The patient achieved remission after the 1st cycle of HiDAC. He also received two cycles of decitabine and arsenic trioxide before being referred to us. The patient relapsed in January 2017 with confirmation of t(11;17) by FISH. On presentation, the patient was asymptomatic and clinical examination did not show any abnormality. CBC and FCI findings are detailed in Table 1. Bone marrow smears showed 22% abnormal promyelocytes and 33% differentiating myeloid cells. Morphological features of the abnormal promyelocytes were similar to case 1. Immunophenotype was similar to case 1 except the CD117 expression in the present case. PCR was used to confirm the presence of ZBTB16-RARA fusion. Baseline Ct value by RT-qPCR was 21.39. The patient was counseled for bone marrow transplantation but was put on palliative care, given non-affordability for transplantation.
Case 5. The patient was a 22-year-old student with a history of fever and body ache for the past 2-3 months. Pallor and tenderness over the rib cage were the only positive findings on clinical examination. Laboratory findings are detailed in Table 1. Peripheral smear examination showed 90% abnormal promyelocytes with strong cytochemical myeloperoxidase positivity. Promyelocyte morphology resembled case 1. In addition, maturing myeloid cells, hypogranular and hypolobated neutrophils were also present (Figure 2, E & F). The patient was immediately started on ATO based on the morphological suspicion of APL. The patient did not develop any bleeding manifestations. The immunophenotype was similar to case 1 and 4, however, unlike those cases, CD56 was dim (Table 1). FISH did not show t(15;17), but the break-apart probe revealed RARA variant translocation. After 11 days of ATO and following confirmation of ZBTB16-RARA fusion by PCR, the patient was shifted to daunorubicin and cytarabine-based induction therapy. Remission was not achieved post induction (Table 1), and the patient was started on consolidation therapy with HiDAC.
|Table 1. Clinico-pathological features of APL cases.|
Case 6. A 35-year-old male was referred to our Institute with complaints of high grade, intermittent fever and back pain for one month. Laboratory features are detailed in Table 1. Peripheral smear revealed 29% abnormal promyelocytes with characteristic morphology and presence of differentiating myeloid series cells (Figure 3). The patient did not develop any bleeding manifestations, in spite of elevated PT and APTT values. Fluorescence in situ hybridization using a dual fusion probe confirmed the presence of PML-RARA fusion in 93% of cells. The patient was classified as high risk as per the Sanz criteria and started on induction therapy with ATO. Post-induction bone marrow examination for response assessment revealed that the patient had achieved morphological and cytogenetic remission. Consolidation therapy included three cycles of daunorubicin and ATRA. The patient is currently doing well and is in maintenance therapy.
Agarose gel electrophoresis for PML-RARA transcript identification revealed a PCR amplicon size of approximately 480bp, which was larger than the expected Bcr3 fusion transcript size (~370bp) (Figure 4). The PCR amplicons were further subjected to Sanger sequencing and revealed an atypical, novel fusion pattern between exon 4 of PML and exon 3 of RARA genes (Figure 4). Interestingly, an additional 40 nucleotides, mapped to intron 2 of RARA, were also coded as part of the mRNA fusion transcript (Figure 4). The addition of exon 4 and 40 nucleotides from intron 2 explains the increase in the size of the Bcr3 transcript type by 111 base pairs (corresponding to the addition of 37 amino acids in the protein).
Thus, this novel Bcr3 variant transcript is in-frame and codes for a fusion protein which is longer than expected PML-RARA fusion transcript product. Cloning of the PCR product into a vector and Sanger sequencing confirmed the above sequencing results (Figure 3). The same plasmid was serially diluted from 106 to 101 copies to be used as RT-qPCR standards. Baseline RT-qPCR revealed high copies (13070) with amplification occurring at a Ct value of 24.7. Post-induction bone marrow evaluation, after 48 days of ATO therapy, revealed fewer than ten copies corresponding to a Ct value of 37.2. Long-range genomic DNA PCR followed by Sanger sequencing identified a breakpoint immediately downstream to exon 4 of PML gene (PML nucleotide no: 74024927) and distal to the 11,789th base (RARA nucleotide no: 40343186) within intron 2 of RARA gene (Figure 4). The first 40 nucleotides of RARA intron 2 (starting from 11,790th base) alone were coded in the protein and were part of this novel fusion transcript.
Case 7. A 26-year-old female was referred with a history of headache and right iliac fossa pain for 2 months. Clinical examination revealed an ill-defined, palpable mass in the right iliac fossa. There was no history or evidence of any bleeding tendency on clinical examination. Abdominal ultrasonography revealed the presence of a 3.3x2.2 cm hemorrhagic cyst within the right ovary. Peripheral smear examination showed 92% abnormal promyelocytes with classical morphology, strong MPO positivity and the presence of Auer rods. The patient was classified as high risk as per Sanz criteria and started on ATO based on the morphological suspicion of APL. FCI revealed typical features of APL; with high side scatter, lack of CD34 and HLA-DR expression, homogeneous bright CD33 and heterogeneous CD13 expression (detailed in table 1). Dual fusion, dual color FISH showed evidence of PML-RARA fusion. RT-PCR for transcript identification yielded an amplicon of ~ 100 bases, higher than that of the expected Bcr1 transcript (390 bases). Sanger sequencing revealed breakpoints within exon 6 of PML and exon 3 of RARA (RARA nucleotide no: 40351910) with the addition of 126 nucleotides, which mapped to the central portion of RARA intron 2 (Figure 5). Therefore the patient was classified as a Bcr2 variant. PML exon 6 was truncated at nucleotide 248, 11 base pairs (bp) upstream of the normal exon 6/intron 6 boundary (PML nucleotide no: 74033403). The insertion of 126 nucleotides from intron 2 can explain the increase in size of the transcript type on electrophoresis. This novel Bcr2 variant transcript is in-frame and codes for a longer PML-RARA fusion transcript product.
Induction therapy was withheld after 38 days due to suspicion of ATO induced hepatotoxicity. However, investigations revealed acute viral hepatitis due to hepatitis E virus (IgM HEV positive). Bone marrow examination for response evaluation revealed complete morphological and cytogenetic remission. RT-qPCR for PML-RARA was also negative. The patient further received consolidation with ATRA, daunorubicin and was started on maintenance therapy. RT-qPCR post consolidation did not detect any PML-RARA copies.
Bcr1 and Bcr3 breakpoints are seen in 55% and 40% of APL cases respectively. Two Indian studies have reported contrasting findings with bcr1 and bcr3 frequencies. Whereas Chatterjee et al. have reported lower bcr1(42.7%) but higher bcr2(14.8%) frequencies; Sazwal et al. (64%) and Dutta et al. (72.7%) have reported over-representation of the bcr3 subtype in Indian patients. The frequency of bcr1 (58%), bcr3 (33%) and bcr2 (5%) subtypes detected at our center was similar to previously published western literature. Breakpoints in Bcr2 leading to the variable (V) isoform are detected in up to 5% patients and have been reported to be more common in the pediatric age group. The Bcr2 variant results from a breakpoint in exon 6 of PML (rarely exon 5) and may show insertion of genomic DNA from RARA intron 2. The size of the inserted RARA intron 2 segments may vary between 3 to 127 nucleotides.[18,19] The loss of distal part of PML exon 6 has been reported to negatively influence the response to treatment.[18,19] We report an adult, Bcr2 variant case showing the addition of 126 nucleotides between PML exon 6 and RARA exon 3 (Figure 5). Our patient responded well to ATO based induction and in spite of complications showed a good response and remains in complete remission.
We also report a unique Bcr3 variant with breakpoint at the junction of exon 4/intron 4 and intron 2 of RARA along with the addition of 40 nucleotides originating from a central portion of RARA intron 2 (Figure 4). To the best of our knowledge, such a case has not been reported previously. Jeziskova et al. also report a unique case with similar breakpoint but with a smaller intronic insertion (9 nucleotides). Our patient showed a slightly delayed response to ATO based therapy, achieving complete molecular remission post consolidation as compared to the patient described by Jeziskova et al., who was in complete molecular remission post-ATRA based induction therapy.
Classical and cytogenetic variant APL’s have distinct natural histories. APL with PML-RARA fusion has a favorable outcome when treated with ATRA or arsenic trioxide. Pharmacological doses of retinoic acid (in the form of ATRA) unbind the N-Cor corepressor from RAR-RXR (RARA-retinoic X receptor) complex and allow transcription of target genes, ultimately leading to myeloid differentiation. The inability of ATRA to dissociate ZBTB16 from the corepressor complex and epigenetic factors such as Polycomb group complexes contribute to the resistance of ZBTB16-RARA APL to conventional therapy. The reduction in ZBTB16-RARA copy number in our cases was determined using relative quantification based RT-qPCR or the delta-delta Ct method. There was no significant change in our treated patients between the baseline and post-induction Ct-values, signifying resistance to ATRA/ATO based therapy. The efficacy of combination therapy with anthracyclines and cytarabine along with ATRA in inducing remission has been previously reported. Rohr et al. describe two cases in which the patients did not achieve remission following induction with ATRA and anthracycline combination but achieved partial remission after reinduction in one of the cases. Even though our case (case 1) was not in remission post induction, no ZBTB16-RARA transcripts were detected by RT-qPCR post consolidation with high dose cytarabine.
Although the clinical features and risk of developing life-threatening disseminated intravascular coagulation is similar to classical APL, ZBTB16-RARA, however, shows distinct morphological features and a lack of response to ATRA/ATO.[5,12] ZBTB16-RARA diagnosis offers a challenge that can be met with a combination of morphological and molecular testing. We report a series of five cases of ZBTB16-RARA associated APL from India with unusual morphological and immunophenotypic features. Cases with atypical breakpoints in PML-RARA represent sporadic events. We also report a unique Bcr3 PML-RARA variant hitherto unreported in the literature.
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