PHA-Induced Peripheral Blood Cytogenetics and Molecular Anslysis : a Valid Diagnostic and Follow-up Modality For Acute Primyelocytic Leukemia Patients Treated With ATRA and/or Arsenic Tri-oxide

  • Baba, Shahid M (Department of Immunology and Molecular Medicine, Sher-I-Kashmir Institute of Medical Sciences) ;
  • Azad, Niyaz A (Department of Immunology and Molecular Medicine, Sher-I-Kashmir Institute of Medical Sciences) ;
  • Shah, Zaffar A (Department of Immunology and Molecular Medicine, Sher-I-Kashmir Institute of Medical Sciences) ;
  • Afroze, Dil (Department of Immunology and Molecular Medicine, Sher-I-Kashmir Institute of Medical Sciences) ;
  • Pandith, Arshad A (Advanced Centre for Human Genetics, Sher-I-Kashmir Institute of Medical Sciences) ;
  • Jan, Aleem (Departement of Clinical Hematology, Sher-I-Kashmir Institute of Medical Sciences) ;
  • Aziz, Sheikh A (Department of Medical Oncology, Sher-I-Kashmir Institute of Medical Sciences) ;
  • Dar, Fayaz A (Advanced Centre for Human Genetics, Sher-I-Kashmir Institute of Medical Sciences)
  • Published : 2016.06.01


Background: Acute promyelocytic leukemia (APML) is characterized by the reciprocal translocation t(15;17) (p22;p12) resulting in the PML-$RAR{\alpha}$ fusion gene. A dual diagnostic and follow up approach was applied including cytogenetic demonstration of the t(15;17) translocation and detection dg PML-$RAR{\alpha}$ chimeric transcripts by molecular means. Purpose: Conventional cytogenetics involving bone marrow is beset with high probability of poor metaphase index and was substituted with phytohemagglutinin (PHA)-induced peripheral blood culture based cytogenetic analysis as a diagnostic & follow up modality in APML patients of Kashmir (North India). Both qualitative (RT-PCR) and quantitative (Q-PCR) tests were simultaneously carried out to authenticte the modified cytogenetics. Materials and Method: Patient samples were subjected to the said techniques to establish their baseline as well as follow-up status. Results: Initial cytogenetics revealed 30 patients (81%) Positive for t(15;17) whereas 7 (19%) had either cryptic translocation or were negative for t(15;17). Two cases had chromosome 16q deletion and no hallmark translocation t(15;17). Q-PCR status for PML-$RAR{\alpha}$ was found to be positive for all patients. All the APML patients were reassessed at the end of consolidation phase and during maintenance phase of chemotherapy where 6 patients had molecular relapse, wherein 4 also demonstrated cytogenetic relapse. Conclusions: It was found that PHA-induced peripheral blood cytogenetics along with molecular analysis could prove a reliable modality in the diagnosis and assessment of follow up response of APML patients.


  1. Ghavamzadeh A, Alimoghaddam K, Ghaffari SH, et al (2006). Treatment of acute pro myelocytic leukemia with arsenic trioxide without ATRA and/or chemotherapy. Annals Oncol, 17, 131-4.
  2. Arnould C, Philippe C, Bourdon V, et al (1999). The signal transducer and activator of trans cription STAT5b gene is a new partner of retinoic acid receptor a in acute promyelocyti clike leukemia. Hum Mol Genet, 8, 1741-9.
  3. Arshad A. Pandith, Mushtaq A. Siddiqi (2012). Burden of cancers in the valley of Kashmir 5 year epidemiological reveals a different scenario. Tumor Biol, 33, 1629-37.
  4. Bennett JM, Catovsky D, Daniel MT, et al (1985). Proposed revised criteria for the classifi cation of acute myeloid leukemia: a report of the French-American-British Cooperative Group. Ann Intern Med, 103, 460-2.
  5. Biondi A, Rambaldi A, Pandofi PP, et al (1992). Molecular monitoring of myl/RAR-a fusion gene in acute promyelocytic leukemia by polymerase chain reaction. Blood, 80, 492-7.
  6. Borrow J, Goddard AD, Gibbons B, et al (1992). Diagnosis of acute promyelocytic leukae mia by RT-PCR detection of PML-RARA and RAR-PML fusion transcripts. Br J Haematol, 82, 529-40.
  7. Carbone R, Botrugno OA, Ronzoni S, et al (2006). Recruitment ro the histone methyltransfe rase SUB39H1 and its role in the oncogenic properties of the leukemia-associated PML retinoic acid receptor fusion protein. Mol Cell Biol, 26,1288-96.
  8. Cassnat B, Zassadowski F, Balitrand N, et al (2000). Quantitation of minimal residual disease in acute promyelocytic leukemia patients with t(15;17) translocation using real-time RT-PCR. Leukemia, 14, 324-8.
  9. Castaigne S, Chomienne C, Daniel MT, et al (1990). All-trans retinoic acid as a differentia tion therapy for acute promyelocytic leukemia. I. Clinical results. Blood, 76, 1704-9.
  10. Chen SJ, Zelent A, Tong JH, et al (1993). Rearrangements of the retinoic acid receptor alpha and promyelocytic zinc finger genes resulting form t(11;17)(q23;q21) in a patient with acute promyelocytic leukaemia. J Clin Invest, 91, 2260-7.
  11. Chen Z, Brand NJ, Chen A, et al(1993). Fusion between a novel Kruppel-like zinc finger gene and the retinoic acid receptor-a locus due to a variant t(11; 17) translocation asso-ciated with acute promyelocytic leukaemia. EMBO J, 12, 1161-7.
  12. Chomienne C, Ballerini P, Balitrand N et al (1990). All-trans retinoic acid in acute promye locytic leukemias. In vitro studies Structure-function relationship. Blood, 76, 1710-7.
  13. David Grimwade, Andrea Biondi, Marie-Joëlle Mozziconacci, et al (2000). Characterization of acute promyelocytic leukemia cases lacking the classic t(15;17): results of the euro-pean working party. groupe français de cytogénétique hematologique, groupe de français d'hematologie cellulaire, UK cancer cytogenetics group and BIOMED 1 Eu-ropean community-concerted action "molecular cytogenetic diagnosis in haematolo-gical malignancies. Blood, 96, 1297-308.
  14. De Botton S, Chevret S, Sanz M, et al (2000). Additional chromosomal abnormalities in pa-tients with acute promyelocytic leukaemia (APML) do not confer poor prognosis results of APML 93 trial. Br J Haematol, 111, 801-6.
  15. Di Croce L, Raker VA, Corsaro M, et al(2002). Methyltransferase recruitment and DNA Hypermethylation of target promoters by an oncogenic transcription factor. Science, 295, 1079-82.
  16. Dilworth FJ, Chambon P (2000). Nuclear receptors coordinate the activities of chromatin re-modeling complexes and coactivators to facilitate initiation of transcription. Oncogene, 20, 3047-54.
  17. Douer D, Santillana S, Ramezani L, et al (2003). Acute promyelocytic leukaemia in patients originating in Latin America is associated with an increased frequency of the bcr1 sub-type of the PML/RARalpha fusion gene. Br J Haematol, 122, 563-570.
  18. Gallagher R, Willman CL, Slack JL, et al (1997). Association of PML/RARa fusion mRNA type with pretreatment hematologic characteristics but not treatment outcome in hematologic characteristics but not treatment outcome in acute promyelocytic leukemia: an intergroup molecular study. Blood, 90, 1656-63.
  19. Ghavamzadeh A, Alimoghaddam K, Rostami S, et al (2011). Phase II study of single- agent arsenic trioxide for the front-line therapy of acute promyelocytic leukemia. J Clin Oncol, 29, 2753-7.
  20. Glass CK, Rosenfeld MG (2000). The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev, 14, 121-41.
  21. Grimwade D (1999). The pathogenesis of Acute Promyelocytic Leukemia; Evaluation of the role of molecular diagnosis and monitoring in the management of the disease. Br J Hematol, 106, 591-613.
  22. Huang M, Ye YC, Chen BR, et al(1988). Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood, 72, 567-72.
  23. Huang W, Sun GL, Li ZS, et al(1993). Acute promyelocytic leukemia: clinical relevance of two major PML-RARa isoforms and detection of minimal residual disease by isoforms and detection of minimal residual disease by retrotrans-criptase/polymerase chain reaction to predict relapse. Blood, 82, 1264-9.
  24. Iqbal S, Grimwade D, Chase A, et al (2000). Identification of PML/ RARalpha rearrange ments in suspected acute promyelocytic leukemia using fluorescence in situ hybridization of bone marrow smears: a comparison with cytogenetic and RT-PCR in MRC ATRA trial patients. MRC Adult Leukaemia Working Party. Leukemia, 14, 950-3.
  25. Jensen K, Shiels C, Freement PS (2001). PML protein isoforms and the RBCC/TRIM motif. Oncogene, 20, 7223-33.
  26. Miller WH Jr, Kakizuka A, Frankel SR, et al (1992). Reverse transcription polymerase chain reaction for the rearranged retinoic acid receptor-a clarifies diagnosis and detects minimal residual disease in acute promyelocytic leukemia. Proc Natl Acad Sci USA, 89, 2694-8.
  27. Mitelman F (1995). An International System for Humann Cytogenetic Nomenclature. S Karger.
  28. Niyaz A Azad, Shahid M Baba, Zafar A Shah, et al (2015). Phytohemagglutinin-induced pe-ripheral blood cytogenetics: a valid means for diagnosis and imatinib therapy monitoring of chronic phase chronic myeloid leukemia patients. J Cancer Sci Ther, 7, 242-248.
  29. Pandolfi PP (2001). In vivo analysis of the molecular genetics of acute promyelocytic leu-kemia. Oncogen, 20, 5726-35.
  30. Pandolfi PP, Alcalay M, Fagioli M, et al (1992). Genomic variability and alternative splicing generate multiple PML/RARa transcripts that encode aberrant PML proteins and PML/RARa isoforms in acute promyelocytic leukemia. EMBO J, 11, 1397-407.
  31. Redner RL, Rush EA, Faas S, et al(1996). The t(5;17) variant of acute promyelocytic leu-kemia expresses a nucleophosminretinoic acid receptor fusion. Blood, 87, 882-886.
  32. Seabrigth M (1971). A rapid banding technique for human chromosomes. Lancet, 2, 971-72.
  33. Shen A-X, Chen G-Q, Ni J-H, et al(1997). Use of arsenic trioxide ($AS_2O_3$) in the treatment of acute promyelocytic leukemia (APML): II. Clinical efficacy and pharmacokinetics in relapsed patients. Blood, 89, 3354-60.
  34. So CC, Wan TS, Chow JL, et al (2011). A single-center cytogenetic study of 629 Chinese patients with de novo acute myeloid leukemia evidence of major ethnic differences and a high prevalence of acute promyelocytic leukemia in Chinese patients. Cancer Genet, 204, 430-8.
  35. van Dongen JJ, Macintyre EA, Gabert JA, et al (1999). Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of mi-nimal residual disease. Report of the BIOMED-1 Concerted Action investigation of mini-mal residual disease in acute leukemia. Leukemia, 13, 1901-28.
  36. Vahdat L, Maslak P, Miller WH Jr, et al(1994). Early mortality and retinoic acid syndrome in acute promyelocytic leukemia: impact of leukocytosis, lowdose chemotherapy, PML/RAR-a isoform, and CD13 expression in patients treated with all-trans retinoid acid. Blood, 84, 3843-3849.
  37. Villa R, Morey L, Raker VA, et al (2006). The methyl-CpG binding protein MBD1 is requi-red for PML- RARalpha function. Proc Natl Acad Sci U S A, 103, 1400-5.
  38. Wang AM, Doyle MV, Mont DF (1989). Quantification of mRNA by polymerase chain reac-tion. Proc. Natl Acad Sci USA, 86, 9717-21.
  39. Warrell RP, Frankel SP, Miller WH, et al(1991). Differentiation therapy of acute promyelo-cytic leukemia with tretinoin (all-t"-retinoic acid). N Engl J Med, 324, 1385-9.
  40. Warrell R.P. Jr, de The H, Wang ZY, et al (1993). Acute Promyelocytic leukemia. N Eng J Med, 329, 177-89.
  41. Wells RA, Catzavelos C, Kamel-Reid S (1997). Fusion of retinoic acid receptor a to NuMA, the nuclear mitotic apparatus protein, by a variant translocation in acute apparatus protein, by a variant translocation in acute promyelocytic leukaemia. Nat Genet, 17,109-113.
  42. Zhong S, Salomoni P, Pandolfi PP (2000). The transcriptional role of PML and the nuclear body. Nat Cell Biol, 2, 85-90. role of PML and the nuclear body. Nat Cell Biol, 2, 85-90.