Liquid Biopsy: A Game Changer

May 29, 2018 0 By S Harachand

The debate is hotting up whether emerging blood-based genetic tests could eventually do away with invasive and troublesome tissue biopsies to determine cancer

The world is awakening to a new era in which detection of cancer is made possible by a simple blood draw at the doctor’s office. Marking a paradigm shift in the manner cancer is diagnosed and treated, a newer modality of malignancy assessment through blood sampling is rapidly gaining ground among clinicians working in the field of oncology worldwide.

Diagnosis and treatment of cancer is usually a drawn-out process and the afflicted often find invasive procedures like repeated tissue biopsies, sort of, a remedy worse than the malady itself.

As hapless patients and care-givers look for new ways of cancer diagnosis which are quicker, easier and less-invasive, testing tumour DNA from the blood sample or `liquid biopsy’, has emerged a probable alternative.

A cutting-edge tool from the stable of genomics, liquid biopsy, employs different approaches to isolate components such as circulating cell-free DNAs (cfDNA) or exosomes from the blood for detecting and identifying cancer.

Facts in fragments

cfDNAs can be found in the plasma as these DNA fragments are shed into the blood as part of certain cellular mechanisms. In case of cancer, this process leads to circulating tumour DNA (ctDNA) in the blood. The technology of liquid biopsy works by isolating these fragments of DNA and identifying specific mutations responsible for malignancies, through just blood sampling.

However, the proportion of ctDNA to the total blood free DNA differs according to the location of cancer, the stage of cancer, the type of cancer and of course, detection methods. ctDNAs will be lower, for example in central nervous system tumours and higher in advanced cancers. ctDNA is identified and quantified using appropriate targets such as KRAS mutation or PIK3CA mutation and various other technologies.

Cell-free DNA analysis is already used for choosing targeted therapies in advanced stages of malignancies. However, there has not been much evidence to support the use of liquid biopsies for early stage cancers till recently.

Now, preliminary data from a large-scale study involving 12,000 patients show that whole-genome bisulfite sequencing (WGBS) assay of cell-free DNA can detect 41 per cent of early-stage (stage I-IIIA) lung cancers and 89 percent of late-stage (stage IIIB-IV) lung cancers at 98 percent specificity. The ongoing Circulating Cell- Free Genome Atlas (CCGA) study has also highlighted the potential of such tests in early stage ovarian and endometrial cancers.

The USFDA approved the first liquid biopsy test in the year 2016 in non-small cell lung cancer (NSCLC) as a companion diagnostic test. The cobas EGFR Mutation Test v2 identifies the mutation status by detecting exon 19 deletion and exon 21 (L858R) substitution in the epidermal growth factor receptor (EGFR) gene. Such mutations are present in approximately 10-20 percent of NSCLC, according to the USFDA .

More clues in ctDNAs?

In addition to ctDNA, tumours also release circulating tumour cells (CTCs) into the blood, which can be enriched using various technologies, for detecting such cells.

Potentially, CTCs have the same scope in cancer management as ctDNA.

However, ctDNA has superseded CTCs in terms of clinical advantage because of the sheer information that ctDNA can provide through next generation sequencing (NGS), in addition to the diagnosis, prognosis, and surveillance benefits.

Earlier studies had mentioned the role of CTCs in the early detection of lung cancer in chronic airway disease patients. One of these studies showed that 50 percent of stage-I lung cancer patients have ctDNA. Similar studies are also going on in patients with inflammatory bowel disease. ctDNA can be detected in up to 73 per cent of the patients with localized cancer, often without simultaneous CTC being present.

Dr Kumar Prabhash, professor, Medical Oncology at Tata Memorial Hospital and editor-in-chief of International Journal of Molecular & Immuno Oncology (IJMIO) confirms that application of liquid biopsy in detection and reassessment in lung cancer is proven without doubt. “Since lung cancer is inaccessible for needle biopsy, it becomes all the more relevant,” he added.

In the same way, other inaccessible tumours such as deep pelvic, pancreatic, and bone tumours can also be diagnosed with CTCs or ctDNA. There are case reports where synchronous versus metastatic malignancies have been identified by detecting a different set of specific mutation.

RNA signatures

Exosome analysis is another modality which seeks to obviate traditional invasive tissue biopsies to diagnose cancer. Exosomes, the small vesicles secreted actively by natural cellular processes, contain all the information from the cell that released it, including RNA and proteins. In the recent annual meeting of American Association of Cancer Research, researchers presented data that breast cancer exosomes isolated from plasma of stage 1 and stage 2 breast cancer patients provided the same RNA signature as that of the matched tumour tissue.

The study used a breast cancer specific marker to isolate breast cancer specific exosomes from the blood and analysed the total RNA content from the samples using RNAseq. The investigators also sequenced RNA from matched formalin-fixed, paraffin-embedded (FFPE) tissue samples from these patients.

Liquid biopsy was able to accurately identify breast cancer RNA signatures from plasma samples along with the breast cancer exosomes from a patient clustered perfectly next to its matched FFPE tissue sample. The breast cancer specific exosomes looked almost identical to the matched tissue. The same breadth and diversity of RNA as seen from the tissue samples has been noted with more than 12,000 different mRNAs and over 1000 lincRNAs.

Enhancing sensitivity

Similarly, it has been demonstrated that the performance of mutation testing can be improved by combining exosome RNA and cell-free DNA, compared to cell-free DNA assays alone.

”You need the exosome compartment to better understand the biology of the cancer,” comments Dr Johan Skog, chief scientific officer at Exosome Diagnostics, a Waltham MA-based developer of biofluid-based molecular diagnostics. “We can also combine the exosome RNA with the cfDNA to look at the mutations on both compartments at the same time. This increases sensitivity.”

Exosomes are released as an active process from living cells, while cfDNA is released from cells dying of apoptosis/necrosis, he added.
In another recently published study, Exosome Diagnostics reported the detection of EGFR T790M mutation in plasma from patients with non-small cell lung cancer (NSCLC).

About 60% of NSCLC patients develop resistance to EGFR inhibitor therapy due to the EGFR T790M mutation. Patients who fail therapy due to this mutation will benefit from treatment with osimertinib. However, performing a repeat lung biopsy to obtain the mutation status is challenging and cannot be done on all patients. Detection of EGFR T790M in a liquid biopsy would solve some of the issues, but it has proven difficult due to low abundance of T790M positive cell-free DNA in blood.

However, utiliising a proprietary technology , the researchers could co-isolate exosomal RNA/DNA and cfDNA (exoNA) in a single step followed by a qPCR based mutation detection assay. Since the process captures both exosomes and cfDNA, it is a considerable improvement upon the current liquid biopsy tests that look at cfDNA alone, according to “Exosome-based Detection of EGFR T790M in Plasma from Non-Small Cell Lung Cancer Patients”, published in the online edition of Clinical Cancer Research, a journal of the American Association for Cancer Research.

Key in precision medicine

This path-breaking technology also enables tracking resistance mechanisms to effectively monitor therapies. Trials are currently underway to identify mutations such as KRAS/NRAS in colon cancer, PIK3CA in NSCLC, BRAF in melanoma and resistant ALK in NSCLC by liquid biopsy. It also has a role in tracing clonal evolution and drug responses to targeted agents, especially by following mutations such as KRAS, NRAS, BRAF, TP53, and PIK3CA.

Clinical trials have established the benefits of liquid biopsy in taking therapeutic decisions. Trials introduced CTC measurements before and after the first cycle of chemotherapy and randomized patients to stay on the same chemotherapy or to switch to another therapy. The trials also confirmed the prognostic significance of CTCs in metastatic breast cancer patients. There are also studies in progress to evaluate the role of CTCs in identifying hormone positive metastatic breast cancer patients who would benefit from early chemotherapy in comparison to hormonal therapy.

 

 

 

“We believe it can replace invasive procedures, or provide
information when tissue biopsy is not available”
Dr Johan Skog
Chief Scientific Officer, Exosome Diagnostics

 

Liquid biopsy vs tissue biopsy

Notwithstanding all this, the question of whether liquid biopsy can altogether replace tissue biopsies — currently the gold standard in assessing different stages of malignancy — remains crucial.

Several studies have established the accuracy of detecting ctDNA as being comparable to that of tumour tissue biopsy. Researchers working in the field are of the view that the technology is powerful enough to transform the expanding precision therapies in different cancers due to its real-time detection of targetable DNA mutations.

”We believe it can replace invasive procedures, or provide information when tissue biopsy is not available (which is frequent),’’ Dr Skog contends, adding that Exosome Tech’s prostate cancer test (EPI) is currently guiding clinicians in selecting correct patients for biopsy using a simple urine sample, helping avoid unnecessary biopsies.

At the same time, oncologists in India seem to have taken a guarded approach to the possibilities of the emerging technology. Many among them, like Dr Prabhash, affirm that liquid biopsy’s role is definitive in detecting and reassessing lung cancer as this form of cancer is inaccessible to needle biopsy.

Liquid biopsy, they feel, is very useful in certain cancers, particularly if the disease has advanced beyond a certain stage. In addition, it can be effectively be used to complement other forms of detection. Genetic analysis of the bio-fluids can obviate repeated tissue biopsies, in cases where it is warranted, they point out.
”In targeted cancer therapy, reassessment is very important and it is very difficult to have multiple biopsies. So I consider liquid biopsy as complementary to the existing techniques even in the accessible cancers,” said Dr BS Ajaikumar, chairman and CEO of oncology-speciality chain Healthcare Global Enterprises.

Evidently, quite a few clinicians believe time has not yet come to reconsider the current gold standard in cancer diagnosis. Dr Narayanankutty Warrier, medical director at MVR Cancer Centre and Research Institute, for instance, holds the view that in the case of accessible cancers, tissue biopsy is still preferred as it always gives better results.

Even though CTCs and ctDNA detection offers great promise, it still needs significant improvement in specificity and sensitivity, they say. Hence it is better limit them to special circumstances including screening and therapeutic re-assessments. More large-scale clinical studies are required to fully recognise how liquid biopsy helps in improving the overall survival rates of cancer patients, they add.

On the other hand, some of the experts are of the view that the initial inhibition towards adopting the technology is nothing but the usual reluctance on the part of the doctors to embrace newer concepts. “For anything new, there will be a resistance as human mind is programmed in such a way that first impulse is to reject the hypothesis, and then look at the data and accept it,” said Dr Anand Babu, associate professor at Kidwai Memorial Institute of Oncology, Bengaluru.

Clearly, there is no consensus yet on whether liquid biopsy is the best option for diagnosing and treating cancers, or whether it should be limited to targeted therapies. Further, it remains a fact that currently we don’t have targeted therapies for all types of cancer. Similarly, the role of liquid biopsy in immuno-oncology is yet to be documented.

More answers would naturally emerge as the technology evolves to perfection. If a simple blood test can obviate invasive tissue biopsies to ascertain cancer, it could certainly be a game changer. But how soon will this breakthrough technology develop into a more efficient substitute for detecting/staging all types of malignancies?

“We will see this type of testing [liquid biopsy] evolve very quickly in the next years,” beams Dr Skog. ”So hopefully, we will have something in the not-too-distant future”

Advantages

Ease of diagnosis: Liquid biopsies avoid invasive procedures, repeated biopsies and overcome the limitation of lacking enough tissue. It is helpful in inaccessible tumours

  • Tumour heterogeneity: It gives more representative information of the tumour and has a potential to detect more targetable/actionable genetic aberrations
  • Prognostication: The amount of ctDNA and the mutational load as well as type of mutations fosters prognostication at the time of diagnosis
  • Prediction of response and effectiveness of anticancer therapy: The quantity of ctDNA, mutational load and specific mutations (appearance of new as well as reversion of old mutations) can be followed up prospectively which can help in therapy decisions
  • Reassessment: To detect residual disease or recurrence and or relapse
  • Better Monitoring: Screening the emergence of new alternative genetic pathways associated with resistance to current anticancer therapy as well as reversion of old active pathways which may warrant change of treatment

Challenges

  • The tumour associated genetic aberrations can be lost or gained over the monitoring period
  • The tumour associated genetic aberrations can be lost or gained in response to treatment
  • The CTCs may detect aberration limited to clonal subpopulation
  • Identification of tumour DNA requires founder genetic aberrations (which are not lost during the progress of cancer) such as APC in colorectal cancer. These aberrations should not be affected by the anti-cancer therapy (passive mutations)
  • Standardization of the testing methods
  • Availability of standardised tests
  • Clinical trials of these tests with survival as endpoints