MOLECULAR PATHOLOGY
Department of Laboratory Sciences
Molecular Pathology
Molecular assays are performed in diverse fields of Medicine, including Oncology, Infectious diseases, Pharmacogenomics and others, providing personalized medical care to patients. At Tata Medical Centre, Kolkata, the diagnosis, sub-classification, management, prognostication and monitoring of treatment response of various hemato-lymphoid malignancies and solid tumors have been made possible by easy-to-understand and implement, customized panel-based molecular assays using laboratory developed tests ( LDTs ) & commercially available tests ; aiding in Precision Medicine. The molecular laboratory is a state of the art facility hosting some of the best equipments for the assays.
Separate facilities are available for Nucleic acids (DNA/RNA) extraction, Polymerase Chain Reaction (PCR), Post-PCR process, Sequencing (Sanger sequencing and Next Generation Sequencing), Droplet Digital™ PCR (ddPCR™)
and Human Leukocyte Antigen (HLA) laboratory, which provide a comprehensive contamination-free environment and reliable assay results.
The nucleic acid extractions are performed manually by using the Trizol reagent as well as by the automated instruments; QiaSymphony® SP (Qiagen, Hilden, Germany) and QiaCube platforms. Our Facility has 5 thermal cyclers which are used for performing PCRs in molecular pathology as well as molecular microbiology: The thermal cyclers available are ThermoFisher® ABI ProFlex, ABI 9700, ABI Veriti, Bio-Rad® S1000 and Bio-Rad® C1000. The agarose gels run for various PCR & Reverse transcriptase PCR tests are documented using the BioRad® Gel Doc XR Plus System.
The quality and quantity of the extracted nucleic acids are checked by the NanoDrop™ 2000 Spectrophotometer, Invitrogen™ Qubit 3.0 Fluorometer and the Agilent™ Biotechnology 4200 TapeStation bioanalyzer. The downstream molecular assays are performed only after the samples pass the required quality / quantity parameters on the above instruments. The department is supplemented with 6 ThermoFisher Bio-Safety Class-II Cabinets, 6 bio-safety hoods, multiple Eppendorf refrigerated / non-refrigerated centrifuges and range of eppendorf micro-pipettes.
A variety of Real-Time PCR (RQ-PCR) and Sequencing (NGS) platforms are available in the Molecular Laboratory; the Real Time PCR instruments being ThermoFisher® ABI 7500, ThermoFisher® Quant Studio™ 3.0, Quant Studio™ 6.0 Qiagen® Rotor-Gene Q and Biorad® QX200™ Digital Droplet PCR. The 4 NGS platforms available at our Sequencing Facility are ThermoFisher® Ion Torrent PGM, ThermoFisher® Ion GeneStudio S5, Illumina® MiSeq and Qiagen® GeneReader NGS.
Liquid Biopsy (cell-free DNA) evaluation is performed using the BioRad® QX200 Droplet Digitial™ PCR. Various liquid & solid malignancies, are diagnosed, prognosticated and monitored using this platform.
Major workload of the laboratory encompasses RQ-PCR for BCR–ABL1 gene fusion transcripts and EGFR gene mutation analysis.The laboratory handles nearly 4000 molecular assays of all types annualy, using all the different platforms installed in the molecular laboratory including the Next Generation Sequencers ( NGS ) equipments.
The HLA laboratory utilizes LifeCodes / LifeScreen Deluxe bead-based Qualitative / Quantitative immunoassay for detecting IgG antibodies against HLA class I and Class II antigens. This is performed on the Luminex® 200™ Multiplexing instrument.
Molecular Pathology at Tata Medical Center, Kolkata, also facilitates outsourcing services for our various medical and surgical oncology disciplines for molecular diagnosis of constitutional syndromes, clinical exome sequencing for rare benign / malignant haematological disorders as well as germline mutation
analysis / genetic counseling services relevant to the clinical profile, helping in the optimal management of patients and their families. The department has Memorandum of Understanding (MoU) with external genomics facilities which provide us with these molecular diagnostics services.
The departmental faculty participates in various extra-mural and intra-mural research projects pertaining to haematological malignancies, solid tumours, and clinical trials, which are funded by agencies like DBT-Wellcome Trust, Tata Trust, ICMR etc.
The department is enrolled in the External Quality Assessment programmes; UKNEQAS, EMQN and ILCPs for maintaining reliable and stringent quality control protocols. The department is a center-for-excellence under DCGI-CDSCO for performing various kit verifications / validations pertaining to solid and liquid malignancies.
TEACHING / TRAINING PROGRSMMES
- Post-Doctoral Fellowship programme in Molecular Pathology (2 Years Duration – 2 positions), which is extendable to a 3rd year.
- Training the Diploma in National Board (DNB) Haematology students (2 students per academic year).
- Yearly Annual Workshop in Molecular Pathology; includes participants from various parts of India ,SAARC and other countries.
- Regular Weekly Inter-lab Training Sessions (WITS) for continued training, education and assessment of Consultants, Fellows and Technical Staff.
- Training Academic sessions to DMLT students.
- Training Visitor – Observers from various institutes in India and abroad (1-3 months duration).
TESTS PERFORMED IN MOLECULAR PATHOLOGY
| Hematolymphoid | Solid Tumors |
| BCR-ABL1 Fusion Gene | EGFR Exon 18, 19, 20, 21 Mutation Analysis (RQ PCR / Sanger) |
| PML–RARA Fusion Gene | Cell free DNA EGFR Mutation Detection Assay (RQ PCR / ddPCR) |
| TCF3-PBX1 Fusion Gene | EGFR C797S Mutation Assay (ddPCR) |
| ETV6-RUNX1 Fusion Gene | c-KIT (Exon 9,11,13, 17) Mutation Analysis ( Sanger ) |
| KMT2A-AFF1 Fusion Gene | PDGFRA (Exon 12, 18) Mutation Analysis ( Sanger ) |
| CBFB-MYH11 Fusion Gene | Microsatellite Instability (MSI) Analysis ( Sanger ) |
| RUNX1-RUNX1T1 Fusion Gene | Combined IDH 1 and 2 Gene Mutation Analysis ( NGS ) |
| PDGFRA-FIP1L1 Fusion Gene | NRAS (Codon 12,13,61,141,146) Mutation Analysis ( Sanger ) |
| Comprehensive Myeloid 69 Gene Panel (NGS) | KRAS (Codon 12,13,61,141,146) Mutation Analysis ( Sanger ) |
| PML-RARA quantification (ddPCR) | RHOA (Exon 2 & 3) Mutation Analysis ( Sanger ) |
| BCR ABL1 Quantitative (e13a2,e14a2,e1a2) | Ion Torrent 50 Gene Cancer Hot Spot Mutation Detection Panel CHPv2 (NGS) |
| BCR-ABL1 Kinase Domain Mutation Analysis | TERT Gene Promoter Mutation Analysis ( Sanger ) |
| Chimerism Analysis | RET ( Exon 10,11,13,14,15 & 16) Analysis ( Sanger ) |
| FLT3-ITD/ TKD (D835Y)Mutation Analysis (Sanger) | MGMT Promoter Methylation analysis ( RQ-PCR ) |
| NPM1 Mutation Analysis (Sanger) | PIK3CA Mutation Analysis (RQ PCR) |
| CEBPA Mutation analysis (Sanger) | BRCA1/ 2 / TP53 / PTEN Mutation Analysis (NGS Panel) |
| c-Kit (Exon 8, 17) Mutation Analysis (Sanger) | TP53 (Exon 2-10) Mutation Analysis (Sanger) |
| NPM1 Quantification (ddPCR) | BRAF Exon 15 Mutation Analysis (Sanger) |
| p190 Quantification (ddPCR) | BRAF V600E Mutation Analysis (ddPCR) |
| MPN Reflex Panel (JAK2 V617F, JAK2 Exon 12, cMPL, CALR) | Outsourced Molecular Tests |
| Thiopurine methyltransferase (TPMT) G460A, A719G, G238C Polymorphism | Clinical Exome Sequencing for Rare Diseases (NGS) |
| MYD88 L265P / CXCR4 Mutation Analysis | Germline Hereditary Cancer Panel (NGS) |
| IGH Somatic Hypermutation / IGHV Mutation Analysis | DPD Mutation Analysis (RQ PCR) |
| T-Cell Receptor Gamma (TCR-G) Gene Rearrangement Analysis | Thalassemia Mutation Analysis |
| IGH Gene Clonality Analysis | Gilbert Syndrome Mutation Analysis |
| TP53 (Ex 2-10) Mutation analysis by (Sanger) | Glucose 6 Phosphate Dehydrogenase (G6PD) Mutation Analysis |
| BRAF V600E Mutation Analysis (ddPCR) | |
| JAK2 V617F Mutation Analysis (ddPCR) |
