The Diagnostic Revolution by the Molecules of Pathology

Dr. Sujay Prasad, Medical Director, Neuberg Diagnostics Neuberg Diagnostics provides best-in-class services in Blood Tests, ECG, Digital X-Rays, Digital Mammography, Sonography, Stress Tests, Spirometry, Audiometry, Eye Checks and Dental Checks, and many more.

Molecules are the Biochemical Foundation of Life
Molecular pathology combines the principles or techniques of molecular biology with diagnostics technology of pathology practice to study these molecules in disease states to provide valuable insights for clinical management and research. It is a highly dynamic and exponentially expanding field that continues to revolutionise our approaches to both diagnosis and therapy.

The relevance of molecular pathology has been demonstrated by the several outcomes of this approach especially by successful landmark therapies targeting genetic alterations including:

• Imatinib treatment of chronic myeloid leukaemia with BCR-ABL gene fusion
• The response of ERBB2 gene amplified breast carcinomas to the anti-ERBB2 antibody trastuzumab
• The efficacy of tyrosine kinase inhibitors erlotinib and gefitinib in the treatment of EGFR-mutated lung adenocarcinomas

" The message of the science must percolate across the boundaries of medicine forming multi-disciplinary team, including pathologists, biomedical scientists and genetic counsellors"

The potential of its applications in clinical diagnostics is ever expanding due to the continuous increase in the number of therapeutic targets and the tremendous demand for companion diagnostics. There are multiple facets of molecular techniques with an ever-widening scope and significance, which necessitates great levels of comprehension with specialized skills for clinical integration.

1. Cytogenetics: The study of chromosomal number and structure by Karyotyping and FISH (Fluorescent In-Situ Hybridization) methods have established role in diagnosis, prognosis and treatment diseases particularly in haematological malignancies.

2. Polymerase Chain Reaction(PCR): PCR-based molecular assays use a pair of priming complementary sequences (oligonucleotide primers) to flank a genetic component(DNA/RNA) of interest, together with unique heat-resistant polymerases(DNA copying enzymes) to amplify and analyse these molecular targets to understand their clinical significance. The development of
specialized PCRs including RT-PCR, Methylation PCR and Digital Droplet PCR will only expand its applications in the study of the molecular pathogenesis for infectious pathogens, genes implicated in cancer and inherited diseases

3. Microarrays:It is a multiplex lab on a chip used for several clinical applications including the classification microbial pathogens, drug and toxic exposures, tumour classification, single nucleotide polymorphism detection, the detection of gene fusions, comparative genomichybridization and gene expression profiling. Micro array-based clinical tests have become powerful tools in the diagnosis and treatment of diseases particularly cancer-related tissue arrays that are bringing together anatomic and molecular pathology.

4. Conventional Sequencing: It includes platforms of Sanger sequencing and pyro sequencing, which have been time, tested but are limited about multi-target assays and still serve a crucial purpose of validating data generated from other methods.

5. Next-Generation Sequencing(NGS): Massively parallel or deep sequencing are related terms that describe a DNA sequencing technology, which has revolutionized several field, including molecular pathology. It can capture a broader spectrum of mutations or genetics targets and provide an option to analyse whole genomic data without any biases.

6. Immunophenotyping:Flow cytometric immunophenotyping offers the sensitive detection of antigens on cell surface and Immuno histochemical(IHC) immunophenotyping involves the process of selectively imaging antigens (proteins) in cells of a tissue section for diagnosis of inherited and malignant diseases. These techniques have extensive applications diagnostic hematopathology and histopathology, as cytogenetic and molecular findings evolve they will bring additional applications of immunophenotyping to field of pathology.

As many molecular techniques, including cell-free DNA analysis (Circulating tumour DNA or Liquid Biopsies), single cell genomics (Circulating Tumour cells), make their expected transitions into the clinical arena, molecular diagnostics will become an integral part of our clinical practice and patient care forming the advanced molecular microscopes of pathology evaluation.

The below two facts highlight both prospects and challenges for molecular pathology in the future.

1. The first sequencing of the human genome with the traditional Sanger technology required approximately 13 years and cost of about $3 billion in 2003 during the completion of the milestone human genome project can now be analysed within a week at a cost close to $1000 by NGS.

2. Currently, even small bench top NGS instruments sequence 3–15 gigabases per day with the reduction in the cost of sequencing enormous amounts of sequencing data creating new paradigms of data analytics and validation for laboratory practice.

The Pathologists of the Future
Pathology is changing, with an exponential magnitude and pace of change the Pathologist must play a crucial role in innovation, making the most of the opportunities offered by technology in all specialties to remain relevant, engaged and contribute to efficient patient care. The pathologist of the future needs to combine morphological methods with practical and theoretical knowledge in genetics, cell biology, biochemistry and bioinformatics to support definitive diagnosis and prognosis.

The main mantra for technology management is to multiplex and collaborate with clinical experts and encourage effective translation of science to clinical care. The path forward is to integrate to accommodate the wide variety of skill sets required to ensure a clinically effective molecular pathology program. The message of the science must percolate across the boundaries of medicine forming multi-disciplinary team, including pathologists, biomedical scientists and genetic counsellors in the laboratory to support the clinical experts in various fields to harness the advances in science and technology and pilot the concept of personalized medicine with the ultimate goal of better patient care.