(NAAC Accredited ‘A’ grade) “Molecular characterization of t(15;17) in FAB classified M3 Acute Myeloid Leukemia patients” (NAAC Accredited ‘A’ grade) Radha Rathee Research Scholar, Deptt. of Genetics, M.D.U. Rohtak, Haryana Dr. Minakshi Vashist Professor and Head, Deptt. of Genetics, M.D.U. Rohtak, Haryana

Leukemia: “White blood” – leucos/aemia 1827 Alfred Velpeau 1845 JH Bennett – patients with enlarged spleens and changes in the colour of the blood – “Leucocythaemia” 1856 Virchow observed the white cells with a microscope – “Leukemia” 28/04/2017

Figure-Development of acute myeloid leukemia. Classification: Acute Chronic Myeloid Lymphoid Figure-Development of acute myeloid leukemia.

Acute Myeloid Leukemia Characterized by immature or undifferentiated hematopeitic cells, i.e. blast cell. Onset is rapid and abrupt Auer rods in M2, M3, M4 Thrombocytopenia Anemia >20% blasts in BM

Clinical Features: Symptoms Pallor and Purpura Skin Infiltration Gum Infiltration Pallor and Purpura Symptoms Skin Infiltration Hyphaema

Insidious nonspecific onset Pallor due to anemia Febrile due to ineffective WBC Petechiae/Pachy skin due to thrombocytopenia Mucus membrane and gum bleed in M4 and M5 subtype

Risk factors for acute leukemia: Ionizing radiation like X-rays, electromagnetic radiations Unsubscribed drugs intake during pregnancy Environmental factors like Chemicals exposure (Pesticides containing benzene, formaldehyde etc.) Genetic history Immunologic factors Viral agents

Translocation (15;17) Acute promyelocytic leukemia (APL or AML-M3) APL is subtype of Acute myeloid leukemia and leads to disseminated intravascular coagulation and death when not diagnosed and treated. More than 99% of APL patients harbor a translocation between chromosomes 15 and 17, which fuses the retinoic acid receptor alpha (RARA) gene on chromosome 17 with the PML gene on chromosome 15. The presence of this translocation is necessary for response to all-trans-retinoic acid and arsenic trioxide.

French American British (FAB) Classification of AML: French American British classified AML into 8 different classes depending upon presence of blast cell and their maturity. M0 -- Undifferentiated AML M1 -- AML without maturation M2 -- AML with maturation M3 -- Acute Promyelocytic Leukemia M4 -- Acute Meylomonocytic Leukemia M5 -- Acute Monocytic Leukemia M6 -- Erythroleukemia (DiGuglielmo’s) M7 -- Megakaryoblastic Leukemia

CYTOGENETICS/WHO Classification: WHO classified AML on the basis of chromosomal anomalies. Different categories are as follows: AML with characteristic genetic abnormalities: which includes translocations [t(8;21)], [t(15;17)], and Inversion [inv(16)]. II. AML with multilineage dysplasia: It includes patients having a prior myelodysplastic syndrome (MDS) or myeloproliferative disease (MPD) that transforms into AML. III. AML and MDS, therapy-related: This category have patients who had prior chemotherapy and/or radiation and subsequently developed AML or MDS. IV. AML not otherwise categorized: It includes subtypes of AML that do not fall into the above categories.

FAB vs WHO: Classifications of AML: WHO criteria Clinical features Morphology Immunophenotyping Cytogenetic features FAB criteria Morphology Cytochemistry

OBJECTIVES: AML PATIENTS To identify patients of AML with the help of FAB classification. To find out the ploidy level in M2 subtype of AML patients. Molecular characterization of structural anomalies in M2 subtypes of AML patients.

TECHNIQUES HEMATOLOGICAL ANALYSIS: 1. Hemoglobin Estimation 2. White blood cell count 3. Leishman’s staining 4. Sudan Black B Staining HUMAN CYTOGENETICS: Lymphocyte culture G – banding Karyotyping MOLECULAR ANALYSIS (RT- PCR): Rna isolation from human peripheral blood Agarose gel electrophoresis

Hematological analysis: 1. Hemoglobin Estimation: 2. White blood cell count:

Blast cells morphology 3. Leishman’s staining 4. Sudan Black B Staining

Whole blood culture and harvesting

46,XX, t(8;21)(q22;q22)

RNA Isolation

Molecular Analysis Isolation of RNA c DNA synthesis Reverse transcript PCR Agose gel electrophoresis Photography (View the gel under UV light)

Outer AML1-primer sequences 5′-AGCCATGAAGAACCAGG-3′ Reverse transcriptase PCR with following primer sequences was conducted. Outer AML1-primer sequences 5′-AGCCATGAAGAACCAGG-3′ Outer MTG8-primer 5′-AGGCTGTAGGAGAATGG-3′ Nested AML1-primer 5′-TACCACAGAGCCATCAAA-3′ Nested MTG8-primer 5′-GTTGTCGGTGTAAATGAA-3′ Translocation t(8;21) was found in 18(9.5%) patients. The translocation t(8;21) alone was found in 14 cases. The FAB subtype of these patients was M2 with SR of 1.25:1. In 4 cases translocation t(8;21) was found together with other abnormalities including trisomy of chr. 8 (n=1), chr. 22 (n=2) and deletion 5 (n=1). Mean values of hemoglobin was 6.4gm/dl (range; 4.2-9.5gm/dl), W.B.C count 7.4X109/L (range; 3.1-16X109/L) and platelet count was 51X109/L (range; 25-78X109/L).

RNA Extraction and quantification RNA was isolated from whole blood using Trizol-method of RNA extraction. Purity of RNA was checked at the OD 260/OD 280. All the samples were found to be in desirable reference ratio 1.9 to 2.0. The purified RNA samples were stored in elution buffer at -800C. Sample ID User Sample Type Timestamp Conc. Unit A (260 nm) A (280 nm) 260 / 280 t(8;21) M2 Radha Rathee RNA 6/19/2014 12:44:38 PM 49.665 ng/μl 0.992 0.480 2.064 Figure- Estimation and quantification of RNA sample of FAB-M2 subtype purity by using Nanodrop method.

RT-PCR amplified product Figure-: Gel image of RT-PCR amplified product for primer PML-RARA. Lane 1-10 amplified product, Lane 11negative control and Lane 12 ladder of 1000bp.

The results of the molecular analysis using RT-PCR of 76 patients were compared with cytogenetics as well as FAB classification. It was found that there were 18 cases of (10 male and 8 female) of AML1/MTG8 [fusion transcript of t(8;21)(q22;q22)] Reverse transcriptase PCR analysis with the help of AML1/MTG8 revealed fusion transcript of t(8;21)(q22;q22) in 18 patients having FAB classification M2 subtype. In two patients (nos. 17 and 18) the t(8;21) was not detected by cytogenetics: patient 17 was karyotyped as t(8;9); patient 18 had a normal karyotype. Similarly, in three patients (nos. 1, 2 and 3), cytogenetic analysis revealed additional chromosomes no. 8 (+8), chromosomes no. 22(+22) and deletion of chr. 5 (-5) in addition to translocation t(8;21). In 12 patients standard cytogenetics did not revealed any other additional chromosomal aberrations.

Translocation detected by Cytogenetics and RT-PCR. Patient Age/Sex FAB/FCM PCR result Cytogenetics result 1 23/M M3 PML/RARa 46,XY,t(15;17)(q22;q12),+22 2 41/M 46,XY,-5,t(15;17)(q22;q12) 3 29/F 46,XX,t(15;17)(q22;q12),+22 4 36/F 46,XX,t(15;17)(q22;q12) 5 39/F 6 38/F 7 42/F 47,XX,t(15;17)(q22;q12) 8 43/F 48,XX,t(15;17)(q22;q12), 9 47/M 45,XY, t(15;17)(q22;q12) 10 45/M 46,XY,t(15;17)(q22;q12)

SUMMARY & CONCLUSIONS One of the molecular-cytogenetic markers associated with a favorable prognosis in AML-M3 is t(15;17). We found t(15;17)/PML-RARA in 4.11% of AML cases.

Future Prospectus The challenging questions with regard to t(15;17) involved leukemogenesis remain as: what are the critical target genes regulated by retinoic acid receptor alpha (RARA) gene on chromosome 17 with the PML gene on chromosome 15. What are the molecular pathways that cooperate with PML/RARA in promoting the oncogenesis of hematopoietic cells? The massive amount of new information about the human and mouse genomes and new technology development will greatly enhance our ability to address these interesting questions.

MAHARSHI DAYANAND UNIVERSITY, Rohtak-124001, Haryana THANK YOU