Liquid biopsies consist of isolating tumor-derived entities present in the body fluids of patients with cancer, followed by an analysis of genomic and proteomic data contained within them. Liquid biopsy has immense potential for early diagnosis, to monitor the intratumor heterogeneity and to serve as a predictive marker for occult metastasis. Despite the potential benefits, the clinical application of liquid biopsy is limited by the lack of specificity and sensitivity, limited clinical validity of bio analyte isolation procedures, and inflated costs. Liquid biopsy in ocular oncology is an emerging topic, which could pave the way for a better understanding of mechanisms for treatment response, resistance, and recurrence in ocular tumors as well as provide specific therapeutic targets to improve life and globe salvage.
Key points
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Liquid biopsy, based on its ability to detect pathogenic mutations accurately and reliably, can aid clinicians in selection of appropriate treatment options and predict the likelihood of globe salvage with current treatment modalities.
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Analysis of the tumor-derived components in the aqueous humor (AH) is an effective technique to disclose molecular alterations and perform genetic analysis.
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Liquid biopsy has the potential to enable precision oncology in the future not only for common eye cancers like retinoblastoma and uveal melanoma but for other ocular tumors as well.
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Liquid biopsy has been analytically validated for research purposes only. Larger prospective multi-center studies are needed for its clinical validity and utility for widespread usage and adoption in ocular oncology.
Introduction
Liquid biopsies consist of isolating tumor-derived entities present in the body fluids of patients with cancer, followed by an analysis of genomic and proteomic data contained within them [ ]. It is a non-invasive or minimally invasive technique for finding tumor components circulating in bio-fluids.
Liquid biopsy is emerging as an alternative approach to analyzing the molecular features of tumors in individual patients. It provides the means to detect and monitor disease progression, recurrence, and response to treatment in a non-invasive way and to potentially complement classical tissue biopsy [ , ].
Liquid biopsy also is a valuable tool for prospective assessment of the genetic heterogeneity of cancer. Traditional biopsies are limited to obtaining tissue from only a portion of the primary tumor, whereas liquid biopsy also reflects the status of any unrecognized secondary or metastatic tumor. It could aid the management of recurrent or metastatic lesions that may exhibit a different molecular profile than the primary lesion [ ].
Liquid biopsy in ocular oncology is an emerging topic, which could pave the way for a better understanding of mechanisms for treatment response, resistance, and recurrence seen in ocular tumors as well as possibly provide specific therapeutic targets to improve life and globe salvage [ ]. In this article, the authors aim to summarize the evolving role of liquid biopsy applications in retinoblastoma (RB) and uveal melanoma (UM), the 2 most common intraocular tumors in the pediatric and adult populations, respectively.
Bio analytes for liquid biopsy
Bio analytes are metabolites that can be harvested from biological specimens such as plasma, urine, serum, and blood and analyzed to derive information about the overall tumor burden as well as the underlying biology of cancer in the body.
Body cells, including cancer cells, release DNA fragments called circulating tumor DNA (ctDNA), which represents a fraction of the circulating cell-free DNA (cfDNA) and cell-free RNA produced through necrosis and apoptosis mechanisms [ ]. In addition, it also is possible to use exosomes, which are subcellular structures encapsulated in the membrane containing proteins and nucleic acids released by tumor cells [ , ]. These components of bio analyte contain information about the molecular and genetic makeup of the tumor and can be used for screening, early detection, and monitoring of response to treatment. Further, neoplastic tumors are in a constant cycle of growth and necrosis. Newer mutations and resistance can develop in this process. With the help of the study of bio analytes harvested in liquid biopsy, new mutations can be screened, and appropriate tailored therapy can be initiated.
The various components of bio analyte that can be used for analysis include
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Circulating tumor cells (CTCs)
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ctDNA
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cfDNA
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Cell-free microRNAs
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Tumor-derived extracellular vesicles.
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Exosomes
The choice of bio analyte for liquid biopsy depends upon the type and anatomic location of the tumor to get the maximum yield of the bio analyte components. Blood makes an ideal candidate and is the most widely used bio analyte for analysis due to the feasibility and convenience of its sampling. However, it has many limitations for its use in ocular tumors in terms of quality yield of cfDNA and other specific bio analyte components. In addition, bilateral ocular tumors express their own specific mutation and with blood as bio analyte, their specific immune cytogenetics cannot be studied.
The aqueous humor (AH) forms in the anterior chamber of the eye, which is a separate compartment from tumors that arise in the retina and the choroid in the posterior chamber. Nevertheless, the AH is enclosed within the same ocular space. Thus, it is an enriched source of eye-specific tumoral genomic information that can be used as a liquid biopsy or surrogate for tumor biopsy for a variety of ocular tumors [ ]. A study has found that AH appears to be a higher yield source of cell-free tumor DNA than the blood when analysis of RB DNA in separate AH and matched blood samples was done [ ].
Characteristics of an ideal liquid biopsy
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Reflects both the status of the primary tumor and the risk or presence of metastasis.
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Monitors the molecular changes that occur as part of the natural history of disease and in response to therapy.
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Is easy to repeat with high sensitivity and specificity.
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Is well tolerated and minimally invasive.
Liquid biopsy in retinoblastoma
RB is the most common primary intraocular malignancy in children [ ]. The diagnosis of RB is mainly based on clinical features and imaging [ ]. Direct tumor biopsy is an absolute contraindication for the fear of extra ocular tumor spread. Therefore, a need exists for proxy markers to allow accurate diagnosis and prognostication to predict the chances of globe salvage in RB.
A significant potential of clinical utility for liquid biopsy exists in retinoblastoma. Bio fluids such as the plasma or AH have been studied to detect ctDNA or cfDNA respectively, to allow for treatment decision-making, monitoring treatment response, and for prognostic counseling [ ]. With the help of liquid biopsy, new mutations can be screened, and appropriate tailored therapy can be initiated.
A liquid biopsy based on blood and plasma samples is not specific to eyes as in a patient with bilateral RB each eye may produce ctDNA that cannot be differentiated in the plasma sample. Thus, AH biopsy has an inherent advantage of being eye specific and can demonstrate inter-eye heterogeneity in cases with bilateral RB [ ].
Further, harvesting the AH from eyes with retinoblastoma has been found to be safe and superior to blood for the detection of chromosomal changes [ ].
The procedure to harvest AH is usually done during part of routine examination under anesthesia [ ]. Certain prerequisites before obtaining the sample are as follows:
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Adequately formed anterior chamber with clear AH.
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Clear media with clear lens and adequate pupil size.
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Normal eye pressure.
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Normal anterior segment on microscopy.
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Non-involvement of the anterior segment of the eye on ultrasound biomicroscopy.
Liquid biopsy can also aid in prognostication of the tumor response to treatment and monitoring of therapeutic intervention. A study has found that somatic copy number alterations and 6p gain in the aqueous humor ctDNA were found to correlate with poorer clinical outcome in terms of 10-fold increased risk of enucleation in such eyes [ ]. Longitudinal evaluation of AH cfDNA has the potential to provide an objective, quantitative way to monitor the therapeutic response and disease burden in RB patients [ , ]. In the enucleated eyes, it was found that 6p gain was associated with aggressive histopathologic features, including necrosis, higher degrees of anaplasia, and focal invasion of ocular structures, demonstrating 6p gain as a potential prognostic biomarker for RB [ , , ].
Liquid biopsy for uveal melanoma
UM is the most common intraocular tumor in adults. Despite good primary tumor control, about half of the patients develop metastasis, which is life threatening [ ]. UM usually presents asymptomatically unless it involves the macula. Clinical examination and imaging are currently the mainstay of diagnosis. Tumor biopsy, including fine needle aspiration cytology, can identify prognostic biomarkers for metastatic UM; however, liquid biopsy may serve as an adjunct. Similar to RB, a study has investigated the role of liquid biopsy using AH in UM eyes and concluded that AH has sufficient cell free DNA to perform genetic analysis [ ].
CtDNA has also shown potential as a liquid biopsy target for screening and monitoring of this disease. Studies have found that UM patients had significantly higher levels of ctDNA in their plasma than patients with uveal nevi, with a strong correlation between ctDNA levels and malignancy [ ]. Studies have also shown that the presence of ctDNA in plasma liquid biopsy in choroidal melanoma patients corroborates with micro metastasis [ , ].
Limitations of liquid biopsy
Liquid biopsy has been utilized in the field of ocular oncology, specially RB and UM, yet insufficient detection sensitivity and consistency and the high cost limit its current widespread utility [ ]. It is known that ocular tumors release exceedingly small quantities of DNA into the circulation thus further limiting the amount of bio analyte available for detailed analysis. In addition, tumor location, laterality, and the presence of inflammation also affect the amount of circulating DNA released. This can lead to high false-negative rates during detection [ ].
Summary and future directions
Liquid biopsy has immense potential for early diagnosis of ocular cancers. In addition, it can be used to monitor intra-tumor heterogeneity and serve as a predictive marker for occult metastasis. Despite the potential benefits, the clinical application of liquid biopsy is limited by the lack of specificity and sensitivity, limited clinical validity of bio analyte isolation procedures, and high cost [ ]. Evolving research and technology in the field of liquid biopsy can combat these challenges, paving the way for its optimal utilization in clinical settings. The application and integration of artificial intelligence to aid in the analysis and interpretation of liquid biopsy is also an evolving area of active research [ ].
Clinics care points
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Liquid biopsy, based on its ability to detect pathogenic mutations accurately and reliably, can aid clinicians in selection of appropriate treatment options and predict the likelihood of globe salvage with current treatment modalities.
- •
Analysis of the tumor-derived components in the aqueous humor (AH) is an effective technique to disclose molecular alterations and perform genetic analysis.
- •
Liquid biopsy has the potential to enable precision oncology in the future not only for common eye cancers like retinoblastoma and uveal melanoma but for other ocular tumors as well.
- •
Liquid biopsy has been analytically validated for research purposes only. Larger prospective multi-center studies are needed for its clinical validity and utility for widespread usage and adoption in ocular oncology.
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