Episode 182. Molecular Pathology with Dr Pranav Dorwal

Molecular pathology provides a comprehensive understanding of disease at its fundamental level and combines molecular analysis with traditional morphological and immunohistochemical methods. The field is continuously evolving as new information becomes accepted into mainstream practice.

With molecular pathology, our understanding of the genetic mutations that lead to disease and our choices of advanced therapies has greatly expanded. It is now uncommon to see a tumour specimen from any organ reported by pathologists without reference to genetic mutations that are known or may be present.

Noting this rapidly expanding field of medicine, I was curious to delve further into the world of molecular pathology, a science often conducted somewhat remotely from the coalface of clinical medicine, yet critically dovetailing into the establishment of treatment algorithms and prognostic modelling. Essentially, I was interested in reviewing some of the basic science at play.

The ten hallmarks of cancer include: genome instability and mutation, resistance to cell death, the ability to sustain proliferative signalling, evading growth suppressors, enabling replicative immortality, inducing angiogenesis, activating invasion and metastasis, reprogramming energy metabolism, tumour-promoting inflammation, and the ability to evade immune destruction.

The precise scientific detail explaining how cells escape normal, healthy biological programming and develop the chemistry that allows these cancer hallmarks to arise is beyond the brief of this podcast. However, it is important to remember that intracellular signalling and cell proliferation have some very important players, which we can briefly review.

Within the DNA housed in a cell’s nucleus, there are genes that both promote normal cell growth and those that function to check or control it. These are called proto-oncogenes and tumour suppressor genes, respectively. In healthy cells, they work in a balanced harmony, with the tumour suppressor genes applying the brakes and providing the checks and balances required to prevent excessive cell proliferation.

Unfortunately, mutations of either proto-oncogenes or tumour suppressor genes disturb this balance of cell metabolism, growth, and function, steering the cell, with its now altered DNA blueprint, toward unregulated proliferation.

Mutations arising through inheritance are referred to as germline mutations; these are present in every cell of the affected individual. The term somatic mutation refers to an acquired error, possibly triggered by epigenetic factors driven by environment or lifestyle such as cigarette consumption, ultraviolet light exposure, or certain dietary choices.

When such mutations affect a proto-oncogene, it is subsequently referred to as an oncogene. We will briefly consider the RAS and BRAF oncogenes, which frequently appear in molecular pathology.

In summary, RAS is involved in so-called upstream chemical signalling and is switched on by incoming signals (ligands) from the external cellular environment. Once activated, RAS triggers an intracellular cascade that ultimately turns on genes involved in cell growth, differentiation, and survival. If RAS genes mutate and become oncogenic, this may lead to the production of permanently activated RAS proteins, inducing overactive signalling within cells.

The three RAS genes in humans, HRAS, KRAS, and NRAS, are among the most common oncogenes in human cancer. Mutations that permanently activate RAS are found in 20 to 25 percent of all human tumours, and specifically in 90 percent of pancreatic cancers.

While RAS genes are found on chromosomes 11, 6, 12, and 1, the BRAF gene, which codes for BRAF protein, is located on chromosome 7. It is involved in downstream signalling and plays a crucial role in regulating cell growth, differentiation, and movement. Mutations alter the activity of various transcription factors, abnormally affecting gene expression and cell growth. BRAF mutations are found in approximately 10 percent of colorectal cancers, up to 50 percent of papillary thyroid cancers, and 27 to 67 percent of melanomas.

You may also have seen other oncogenes in reports, such as MYC (which encodes a transcription factor), EGFR (Epidermal Growth Factor Receptor, involved in cell growth and survival), and HER2 (Human Epidermal Growth Factor Receptor 2), a protein that regulates cell growth and division. Amplification of HER2 is a hallmark of some breast and ovarian cancers.

This knowledge is significant, as several oncological treatments including JAK inhibitors (ending in nib) and monoclonal antibodies target these abnormal protein pathways or cell receptors. A single mutation is all that is required to activate an oncogene.

Conversely, tumour suppressor genes are involved in a variety of functions, including repairing misaligned DNA segments where opposite strands become mismatched (as in Lynch syndrome gene mutations), cell cycle control, apoptosis promotion, and telomerase regulation.

Mismatch becomes more common with ageing as telomeres shorten, and the repair process is referred to as mismatch repair. A mutated mismatch repair gene can no longer carry out this function, leading to abnormal protein synthesis and maladaptive cellular processes. As we will learn in this podcast, these gene abnormalities can be detected in tissue specimens after molecular pathologists apply analytical methods such as immunohistochemistry staining.

The terms mismatch repair proficient (normal genes detected, mutation not present) and mismatch repair deficient (mutation present) often appear in tumour reports, especially in relation to colon cancer.

The BRCA1 and BRCA2 genes, as well as the Lynch syndrome genes (MLH1, MSH2, MSH6, and PMS2), are well-known tumour suppressor genes. When mutated, they dramatically increase the risk of developing breast, ovarian, and prostate cancers (BRCA), as well as colon, uterine, urogenital, pancreatic, and other cancers (Lynch).

While these mutations are generally inherited, they may also arise spontaneously or through epigenetic silencing. As there are two copies of each gene, and only one is generally inherited in familial cancer syndromes, a somatic mutation of the other copy must occur before tumour suppressor activity is lost. This is the two-hit hypothesis, proposed in 1971 by Knudson, which explains tumour development as we age. Interestingly, inheritance is associated with a high rate of somatic mutation in the second gene copy.

The term methylation is sometimes included in pathology reports. Methylation of particular amino acids within a gene, specifically CpG (cytosine-phosphate-guanine) sites, possibly driven by epigenetic factors, leads to dysfunction in DNA repair processes. The resulting mismatch contributes to failed tumour suppression.

As noted, this was a simplified overview of a fascinating and complex branch of science.

To discuss this further, I would now like to introduce Dr Pranav Dorwal, a molecular and anatomical pathologist who works in the Departments of Diagnostic Genomics and Anatomical Pathology at Monash Health. Dr Dorwal is an examiner for molecular pathology, an active researcher, and has more than 60 publications to his credit.

He has worked in the molecular pathology services at MD Anderson Cancer Center (Houston, TX, USA) and Memorial Sloan Kettering Cancer Center (MSKCC, NY, USA). Pranav undertook a visiting fellowship at the Australian National University (ANU), Canberra, and was awarded the Chancellor’s Gold Medal during his Diploma in Clinical Pathology.

Please welcome Dr Pranav Dorwal to the podcast.

References:

Dr Pranav Dorwal – www.monashhealth.org | www.genomicdiagnostics.com.au

Oncology at a Glance, Graham Dark, Wiley-Blackwell

www.pmc.ncbi.nlm.nih.gov