Lung cancer is the most common cause of mortality from cancer worldwide. Despite recent developments on early diagnosis and tumor molecular profiling, this disease remains the most aggressive type of malignancy with only less than 20% 5-year survival rate.1-4 Among the two types, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), the latter has a higher incidence rate especially among Asian patients which has prompted tumor gene profiling a necessary step at the time of diagnosis. For 5 years, our institution has detected 31.37% positive cases of EGFR tissue mutation (56% were female) and 7.84% positive cases of anaplastic lymphoma kinase (ALK) mutation.
Over the years, several advancements in tissue biopsy were invented to screen pulmonary malignancy. However, there are still certain important issues that prohibit prompt establishment of diagnosis. Among these are: intratumor heterogenicity, tumor accessibility, increased rates of morbidity (pneumothorax), poor health status of patients and the long waiting time before cytopathology results are released.5
The standard management for early stages of NSCLC is surgery.2 Following treatment of early stages of lung cancer, a series of CT scans are done every 3-6 months to monitor disease progression.6 While surgical resection offers a long-term survival, tumor recurrence occurs in 50% of cases.7,8
Meanwhile, for late stages of lung cancer, tissue biopsy is necessary for tumor molecular profiling to identify biomarkers necessary for treatment. Tumor recurrence and tyrosine kinase inhibitor (TKI) resistance are also identified through tissue analysis. The problems however, rise when the biopsy risk is too high for weak patients, lung nodule location is too deep and tumor tissue is inadequate for molecular profiling. The goal is to perform the least invasive yet effective diagnostic procedure that could provide information equal to the data offered by tissue biopsy.
Liquid biopsy is a cutting-edge technology that allows screening and monitoring of tumor gene profiling through investigation of cell-free DNA (CfDNA) in whole blood. This test only requires venous blood but provides wide information on EGFR and gene analysis.4
The aim of this study is to identify and evaluate the role of liquid biopsy in lung cancer diagnosis, monitoring, and treatment.
Between September 2016 and December 2016, we retrospectively collected and reviewed the medical records of thirty-five patients who were suspected or diagnosed of lung cancer, then underwent serum epidermal growth factor receptor (EGFR) or liquid biopsy for further investigation. We also reviewed the information of subjects previously diagnosed with lung malignancy and receiving treatment who came for investigation of possible recurrence or resistance. Data gathered were patient age, gender (male or female), smoking history (smoker, previous smoker or never smoked), final diagnosis established through tissue biopsy or surgery, tissue gene mutations (EGFR and ALK) and EGFR liquid biopsy results. We evaluated the accuracy of liquid biopsy by comparing the results with tissue pathology reports and final diagnosis.
The sensitivity (true positive rate) and specificity (true negative rate) were computed by using this formula:
From September to December 2016, a total of thirty-five patients underwent an EGFR liquid biopsy test. The final diagnosis revealed 18 subjects (51%) were cancer free and were given the following diagnoses: tuberculosis and nontuberculous mycobacteria (NTM): ten (56%); other infections: four (22%); benign nodules: three (17%) and squamous metaplasia: one (6%) (Table 1). Meanwhile, seventeen subjects
(49%) were positive as pulmonary malignancy. Among the seventeen, fourteen (82%) were diagnosed as adenocarcinoma, one (6%) anaplastic giant cell, one (6%) carcinoid tumor and one (6%) of thyroid cancer, lung metastasis (Table 2).
It is important to note that none of the cancer patients were active smokers. There were only five (29%) previous smokers and twelve (71%) non-smokers. The presence of tuberculosis with lung malignancy was also observed on 35% of cases. For the fourteen cases of Adenocarcinoma, four were in the early stages and ten in advanced stages (Table 3). All four early stages and one patient in stage IIIA underwent surgical procedure and tissue specimens were sent for EGFR and ALK analysis. Three cases were positive for tissue EGFR exon 21 and two cases were positive for EGFR exon 19. One case underwent liquid biopsy test pre-surgery which detected EGFR exon 21 L858R mutation followed by another liquid biopsy test four days post-surgery with negative results. The other four cases underwent EGFR blood test only after surgery which reported negative EGFR mutation (Table 4).
For the advanced stages who underwent chemo, radio and/or tyrosine kinase inhibitor (TKI) therapy, four subjects underwent liquid biopsy test prior management which reported three cases of serum EGFR exon 19 detection, confirmed by tissue EGFR diagnosis and an isolated case was negative for EGFR liquid biopsy but was positive for tissue EGFR exon 19.
Two advanced stages were on long term treatment (2.5 years and 1.6 years) and were investigated for possible recurrence. One case was positive of serum EGFR exon 20 T790M and EGFR exon 21 L858R developing resistance from the first line of TKI therapy. Bronchoscopy was not performed, and CT guided biopsy was unsuccessful due to the size and location of the lung nodule. The other case was negative for serum EGFR after 1.6 years of TKI treatment. Another subject was found negative for serum EGFR mutation 59 days on chemo, radiotherapy and TKI treatment when she was positive for tissue EGFR exon 21 upon diagnosis.
One advanced case was monitored during diagnosis and thirteen days after first day of TKI therapy. The subject was positive for serum EGFR exon 19 as well as tissue EGFR exon 19 then negative of serum EGFR thirteen days on treatment.
The last advanced stage of adenocarcinoma was negative for serum EGFR after long term chemo therapy but later the patient deteriorated and was moved to palliative care instead.
The sensitivity of liquid biopsy (true positive rate) is 100%, specificity (true negative rate) is 95.5%, false positive rate is 0% and false negative rate is 14%.
NSCLC is a challenging disease to diagnose and manage. This requires tumor biopsy for tissue gene mutation analysis which is not without risk and complications. For the past decade, our institution has managed to keep up with the advancements of interventional pulmonology, being able to perform the following investigative and treatment measures: Fiberoptic bronchoscopy (FOB), brachytherapy, electrocautery, argon plasma coagulation, autofluorescence, endobronchial ultrasound (EBUS) guided sheath (GS) and transbronchial needle aspiration (TBNA), electromagnetic navigation bronchoscopy (ENB) and fiducial marker. All of these are invasive, requiring a bronchoscope entering the airways which necessitates the patient to be under anesthesia (local or IV sedation). While all of these methods have proven effective in their own ways, there are still barriers when handling cases of NSCLC.
In a recent article, Dr. Marius Ilie and Dr. Paul Hofman,9 discussed the advantages of liquid biopsy. They mentioned some of the common concerns pulmonologists encounter during lung biopsy and the role of liquid biopsy to conquer these barriers. We further elaborated our experience on these hindrances.
Intratumor genomic diversity greatly affects how one responds and reacts to therapies when there is progress of malignancy and/or treatment resistance. In a study conducted by Dr. Zhang et al.10 wherein eleven tumor regions from surgery where investigated, a total of 7,269 mutations and 7,026 (97%) somatic mutations were seen on their experiment. This means that when intratumor heterogenicity is possible, pulmonologists should consider a given tumor like a tree structure having a trunk or main mutation that is present in all regions while the branches have different mutations on separate parts of tumor.10
Failure to recognize these mutations may highly contribute to the development of TKI resistance by starting therapies that do not perfectly suit the presence of mutations within the tumor. Liquid biopsy is a source of fresh tumor-derived material from CfDNA of the lung tumor from whole blood. This detects mutations from circulating tumor DNA (ctDNA) rather than just investigating a part of tumor acquired through biopsy.10 This system gives the pathologist a bigger picture of the mutations present in the lung tumor, and therefore mutations such as EGFR T790 can also be detected.
There are 10-30% activating EGFR mutations found in NSCLC patients.11-14 These activating EGFR mutations are related to effective rates to EGFR-TKI therapy. One research with combined results from 21 studies from the years 2004 to 2006 showed a response in 210 out of 268 patients (78%).15-16 However, a secondary mutation of T790 has been reported to have acquired resistance, therefore making its presence of important clinical relevance.17 In our study, one case after 2.5 years of first line TKI therapy was identified with EGFR exon 20 T790M and EGFR exon 21 L858R through liquid biopsy. Tissue diagnosis was not possible as the lung nodule was located deep within the lung and even CT guided needle biopsy was unsuccessful. This shows how liquid biopsy could fill the gap when tissue biopsy is not possible for advanced cases of lung cancer. Upon knowing the presence of T790 mutation, treatment was immediately revised without having the need to wait for tissue diagnosis and tumor gene analysis.
A study by Dr. Wong, et al18 stated that 31% of cases have hard to reach lung nodules. In some cases, there is an insufficient amount of viable tissue which limits information for tumor gene analysis. Performing liquid biopsy in these scenarios would have prevented unnecessary repeat biopsy and or surgery.
We have proven that tumor gene analysis can be obtained not only through tissue biopsy but through liquid biopsy as well. Our test’s specificity is quite high so a positive result rules in EGFR mutations. If it’s negative, then we still proceed to do the biopsy. In our study, there were five adenocarcinoma cases that underwent surgery. One subject was positive for serum EGFR exon 21 pre-surgery confirmed by tissue gene analysis post-surgery. Four days after the operation, the subject was negative for liquid biopsy. Meanwhile, four cases presented negative serum EGFR with varying number of days post-surgery. This proves that the role of liquid biopsy in the early stages of lung cancer includes: early detection by blood screening; timely surgical removal of tumor; and avoidance of recurrence through a series of serum EGFR monitoring. This test avoids repetitive radiation exposure by chest x-ray or CT scan and prevents unnecessary invasive procedures such as bronchoscopy.
There were nine cases of advanced stages of lung cancer. Three were positive for liquid biopsy EGFR exon 19 upon diagnosis, confirmed by tissue gene analysis (EGFR exon 19) through lung biopsy. One case reported resistance with EGFR exon 20 T790M and EGFR exon 21 L858R result from liquid biopsy. Tissue biopsy was unsuccessful due to the size and depth of the nodule. From these cases, we have validated that the role of liquid biopsy for advanced stages includes: diagnosis through serum collection; evaluation of TKI therapy resistance; examination of possible disease progression; and a good alternative for tissue biopsy when the tumor is not accessible and or the patient has poor health status.
Another function of liquid biopsy is the evaluation of affectivity of chemo and TKI therapy. In our study, three cases with varying number of days when blood extractions were performed, revealed negative serum EGFR. All three patients were initially positive of tissue gene mutation, underwent treatment then showed negative serum EGFR analysis. One of these three cases was too weak to undergo tissue biopsy.
One important advantage of liquid biopsy is the prompt turn-around time from serum collection to release of results which provides clinicians information for timely execution of treatment. One advanced stage patient underwent liquid biopsy prior to lung tissue biopsy which revealed positive serum EGFR mutation. Instead of a two-week waiting period for tumor tissue gene mutation results, TKI therapy was immediately executed once final diagnosis of adenocarcinoma was established through tissue biopsy. This provided our subject an extra two-week start of therapy.
Although liquid biopsy has shown its importance in all stages and phases of lung cancer, this should not replace tissue biopsy as the gold standard for lung cancer diagnosis. To prove our point, one of our advanced stages reported a negative serum EGFR result but later revealed a positive tissue EGFR post-surgery. Although this can be an isolated case, one must still consider that while serum gene profiling is promising, tissue biopsy should be maintained as the benchmark when considering lung cancer as a possible diagnosis.
Another disadvantage of liquid biopsy is that the ctDNA collected from the serum could be from shedding of primary or metastatic tumor which may provide false information. This means that for late stages with metastases, radiographic imaging, tissue analysis and liquid biopsy as a combination should be highly considered.
While our study is still in its premature phase, the authors would like to express how within the span of four months this blood test was able to secure its purpose in all stages of lung cancer. This is just an initial study with substantial results but the authors will commence further investigation and research.
Liquid biopsy is a minimally invasive procedure that provides information about genetic alterations in patients with pulmonary tumors. This test is a significant development in lung cancer diagnosis, management and monitoring. This presents accurate data that could compliment radiographic imaging, tissue analysis for diagnosis, early execution of treatment, follow-up evaluation post-surgery, TKI therapy efficacy monitoring, TKI resistance screening and determination of cancer recurrence. This crucial test may improve patient outcomes by faster treatment decisions, avoiding invasive tests such as tissue biopsy or surgery and anticipate disease progression even before radiographic images detection.
Although liquid biopsy will not replace tissue studies, this test is an important tool that will bring favorable changes in overall management of suspected or diagnosed patients of lung cancer.