Despite the availability of technologies such as quantitative PCR (qPCR), digital PCR, and next-generation sequencing (NGS) to guide personalized treatment of several cancers, they are not yet an option for millions of patients worldwide. In this interview, our Senior Marketing Manager, Dr. Deepak Tripathi, discusses this bench-to-bedside gap with Dr. Anuradha Chougule, whose persistent efforts have helped establish affordable genomic testing for cancer patients in India. Dr. Chougule currently serves as Professor Chair of Excellence for Research in Cancer Genetics at the Maharashtra University of Health Sciences (MUHS) in India.
You have been working in the translational cancer field for a long time. What motivates you to fight cancer, and what led to your interest in oncology?
My interest in oncology goes back to my PhD days at the Bhabha Atomic Research Centre, where I studied oxidative stress and how it causes DNA mutations. That inspired me to continue working in the cancer biology field. DNA mutations occur when our body’s DNA repair system gets saturated and goes beyond the repair limit. During my research work, the focus was identifying on individual Reactive Oxygen species like Superoxide radical, Hydroxyl radical, NQO1, Cytochrome P 450.
When I moved into the cancer diagnostic field, I wanted to see how we could make tests more cost-effective. We realized that patients from low socioeconomic groups in the Tata Hospital could not afford even a 2000- or 5000-Rupee test. So, we started with the BCR-ABL and PML-RARA testing in leukemia.
We started offering the BCR-ABL real-time PCR-based test for 900 Rupees when imatinib treatment was made available. After that, I established several bench-to-bedside tests in the department, including PML-RARA, EGFR, EBV and DPYD tests. In these cases, Sanger Sequencing was very useful for looking at the various exonic mutations. For instance, before starting a patient on 5 fludarabine, DPYD homozygous/heterozygous mutation status is determined by Sanger Sequencing. If it is heterozygous, physicians adjust the dosage of 5 fludarabine, but if it is homozygous, they have to look for another option. This was at a time when NGS was not available, and therefore, Sanger sequencing was the go-to method.
Later, we designed a 10-gene lung cancer panel with NGS, which included EGFR, KRAS, BRAF, EML4-ALK, RET ROS, MET exon 14 skipping PIK3CA, and AKT mutations. This information helped medical oncologists to determine which mutations to target. Developing these affordable bench-to-bedside tests was a big motivation for me then. My current focus, however, is more on the research side. I want to work on Genome editing in cancer research.
It’s encouraging to see how scientists like you strive to close the bench-to-bedside gap. Do you see any benefits of using qPCR and digital PCR in translational cancer research?
Talking about the benefits of qPCR, when you are performing the BCR-ABL quantification, we look for the log reduction value at the 3-month interval when the patient is on imatinib treatment. You can monitor the disease better with the log reduction value with qPCR. With a 10-log reduction, you reach a stage where you just need to monitor the disease every year, but again, we need to see whether there is an increase in the BCR-ABL copy numbers. However, unlike qPCR, digital PCR helps to absolutely quantify the copy numbers.
Another thing I want to emphasize is liquid biopsy and cfDNA (cell free DNA) detection. For example, with gefitinib treatment, you can do a longitudinal copy-number monitoring to adjust the dose or continue the current regimen. Both qPCR and absolute quantification with digital PCR have been very useful in translational research.
Could you elaborate on why scientists like you should use qPCR or digital PCR complementary to NGS in a translational setup?
In a translational setup, NGS definitely has its advantages; there is no doubt about that. The study of whole exome, whole genome, and transcriptome is more useful in translational research than in the clinic. Unlike NGS, with qPCR and digital PCR you can easily monitor the disease with single gene markers. Also, after NGS determination of low frequency and rare mutations, we have to go back to qPCR or digital PCR to validate and determine the copy numbers. So, I would say they are complementary to each other.
Would you then say that the complementary use of qPCR, digital PCR, NGS, and Sanger sequencing can give us the complete picture of what is happening inside a tumor?
For the reflex test, you can say that. However, qPCR is much more helpful for orthogonal testing in molecular diagnostic departments.
Are qPCR and digital PCR more sensitive than NGS?
NGS reporting is tricky because it depends upon the coverage, variant allele frequency (VAF), and the initial tumor content. Only if you have more than 30% tumor content, you can perform NGS with good depth coverage to get the true VAF. But if the tumor content is lower, between 30% and 20%, you can perform Sanger sequencing, and if it is between 20% and 10%, a highly sensitive approach like real-time PCR is more useful. Lastly, for less than 1% tumor content, absolute quantification with digital PCR would be the most reliable.
Can you comment on the increasing role of genomics in personalized medicine?
With the increasing use of genomic technologies in personalized medicine, we are seeing a paradigm shift from chemotherapy to personalized medicine. Earlier, it was ‘one size fits all’. With a precision medicine approach based on comprehensive genomic profiling, the whole spectrum of mutations can be considered before treatment. This whole-genome profiling approach is one of the advantages of NGS over qPCR. For instance, there are targeted therapies for the presence of EGFR exons 19 and 21 mutations, as well as KRAS G12C mutation. However, the combination of EGFR and KRAS mutations would indicate poor prognosis. Hence the combination of actionable driver mutations, predictive markers, and prognostic markers becomes very helpful in guiding treatment.
What challenges do you face in delivering patient care?
The main challenge, especially in India, is that despite having the information about the actionable driver mutations, we are unable to use it to deliver patient care. This is because many of the FDA-approved targeted therapies are not yet available in India. So, at this point, gaining information about actionable mutations is only useful from a research data point of view.
What are the challenges, particularly with respect to reproducibility and sensitivity that you face while doing cancer research?
A highly reproducible test is critical for cost-effectiveness. For example, in qPCR, if you have to perform the test in triplicates for reproducibility, it won’t be a cost-effective test. Hence, reproducibility is considered when choosing the primer probes or the whole kit.
Sensitivity is an important consideration for FFPE tissues. From FFPE tissue blocks, due to crosslinking, we do not get very good RNA. This makes it very challenging to detect fusion genes using NGS. In these cases, particularly when you have less than 10 percent tumor content on the FFPE tissue, qPCR or digital PCR are more suitable.
As a liquid biopsy expert, to what extent do you think liquid biopsy complements radiographic imaging in the reduction of early recurrence of lung cancer?
There are several challenges in liquid biopsy. The success of the procedure depends heavily on how and when the blood is collected, transported, plasma adequately separated and stored. However, despite these challenges, liquid biopsy is a promising tool in molecular biology, particularly in cancer biology. It is not always possible to get a repeat tissue biopsy from patients. This is where liquid biopsy really comes into the picture. And liquid biopsy is not just limited to plasma biopsy; if there is metastasis, there might be an accumulation of ascitic fluid or pericardial fluid, pleural effusion and CSF which can be used for cell-free DNA (cfDNA) extraction and genome profiling. Urine and saliva are other types of true non-invasive samples for liquid biopsy testing. I believe that these non-invasive liquid biopsies are going to be very useful for the patients. Also, even if we can detect only one CTC per 7 ml of blood, this could indicate that the patient might develop metastasis in the future, making liquid biopsy really useful as an early detection method.
What are your thoughts on immunotherapy?
Yes, there is a future for immunotherapy – immune checkpoint inhibitors and targeted therapies. With targeted therapies, we target the mutated DNA, whereas with immune checkpoint inhibitors, we target the T-cell receptors. Identifying PD-L1, a programmed death ligand marker, and CTLA-4, a T-cell receptor marker, at the earliest is important. Seeing the expression of these proteins can guide treatment with immuno-oncology (io) therapies. Apart from these, tumor mutation burden (TMB) is a critical biomarker. With TMB, higher number of mutations detected means more neo-antigens on the cell surface receptors, and therefore better chances that the patient will respond to immunotherapy. High TMB levels (≥ 20 mutations /Mb) correspond to a 58% response rate to ICIs while lower TMB levels (≤ 20mutations /Mb) reduce response to 20%. And the same goes with MSI or microsatellite instability, where if a patient is MSI stable, then he/she would not be able to go for immunotherapy. But for MSI-high patients, immunotherapy is a suitable option. So, nowadays, clinicians are looking at multiple treatment strategies – chemo plus ICI, chemo plus TKI, or the TKI plus ICI and how they can reduce toxicity for these patients. Besides, the relevance of these biomarkers could vary with the cancer types. TMB is not a very relevant biomarker in lung cancer but is relevant in colorectal and GI cancers. Another challenge is the lack of consensus in the community on whether TMB should be considered as a biomarker or how many mutations should be considered as high TMB. Unlike TMB, PD-L1 status can be determined by immunohistochemistry, and we can rely on these reports to recommend io therapy.
How important is a multidisciplinary collaborative approach in your day-to-day work? How do you think this would benefit patients and the wider society?
I participate in molecular tumor board meetings. This is where we discuss all reports that come in. Scientists play an important role in these meetings, helping clinicians understand and interpret the reports – what is the significance of it, and which signaling pathways are relevant – which ultimately helps make them make better decisions for the patients.
Medical oncologists play a vital role in this multidisciplinary approach. They bring scientists into the picture, particularly to develop NGS-, PCR- or Sanger sequencing-based tests to guide the use of new targeted therapies. Also, cost is a critical consideration here. For instance, for NGS, you need a very costly instrument. This would mean, a 10-gene panel would cost 10,000 to 12,000-Rupees per patient, making it unaffordable for many patients. So, I would recommend going for a minimal small-gene panel, a very useful yet affordable option for patients.
Do you think Applied Biosystems has a robust portfolio for comprehensive genetic analysis? How do they help you achieve your research goals?
Our molecular biology department uses instruments and other solutions from Applied Biosystems. Your end-to-end solutions – from pipettes, centrifuges, and freezers to digital PCR, Sanger sequencing, and latest NGS platforms like the [Ion Torrent Genexus System] with whole-genome and whole-exome sequencing capabilities – are quite robust. Also, we have never had repeated instrument breakdowns and there has always been very strong support from the Applied Biosystems engineering team. I also wish to try out the new microarray platform for gene expression analysis.
I am also very much fascinated with the Thermo Fisher CRISPR-Cas9 [technology]. I recently used it in an in vitro experiment for a DST DBT grant I submitted on lung cancer organoid. I do have one concern: it would be beneficial if Applied Biosystems offers an open platform where you can use any other kit. The technology has improved greatly with the newer versions of Ion Torrent sequencing platforms – the [Ion GeneStudio™ S5 System], [Ion GeneStudio S5 Plus System], [Ion GeneStudio S5 Prime System] and the Genexus. These milestones of Thermo Fisher would indeed help us achieve higher throughputs.
We’ve discussed enough science, let’s get personal. Tell me something about your hobbies, what do you do in your free time?
I love playing Carrom. I’ve represented the Tata Memorial Hospital under the Bombay 1 group under DAE multiple times and won. It is a very geometric based game that helps you develop your concentration and skill. Besides, I cook and enjoy reading, particularly books written by Robin Cook.
This interview has been edited for length and clarity.
Learn more about Applied Biosystems solutions for cancer research at thermofisher.com/abcancerresearch.
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