In the past decade, the next-generation sequencing (NGS) technology has changed the field of oncology, leading closer to precision medicine. Precision medicine in oncology aims to identify an individualized treatment plan based on specific molecular or cellular features (genomic or protein changes) in a patient’s tumor(1). In other words, precision oncology in cancer uses biomarker testing to guide oncologists on the most beneficial therapy for an individual patient. Treatments, therefore, are genomically informed and individualized, based on the patient’s genetics, metabolic profile, environment and habits(2). Today, both common and rare cancer can be targeted by specific therapies to improve clinical outcomes in patients. Constant reduction of NGS-associated costs has allowed the characterization of the cancer genomic complexity, identifying “actionable” targets and “driver” mutations responsible for tumor growth and progression. Both FDA and EMA approved biomarker-matching targeted drugs and routine molecular pathology testing(3). Furthermore, the combined use of NGS gene panels that expand the number of genes that can be simultaneously analyzed along with immunohistochemistry-based techniques increase the possibility of finding a potentially beneficial targeted therapy. Successful examples include anti-HER2 therapy for HER2/neu overexpressing gastric or breast cancer patients(4-9), the use of vemurafenib for BRAF V600E mutant metastatic melanoma patients(10-12), or PARP-inhibitors for BRCA-mutant advanced ovarian(13) metastatic breast(14,15) and metastatic pancreatic adenocarcinoma patients(16,17). Moreover, next-generation sequencing has allowed patients worldwide to receive personalized treatments, particularly for cancers that have no approved treatment options. Based on this approach, off-label therapies may be suggested according to the biomarkers identified in a tumor regardless of the cancer type or where cancer started in the body.
Despite these promising results, the implementation of precision medicine approaches in oncology is far behind the large-scale molecular data and information available to date. Translation of big genomic data into clinically actionable information has been primarily slow due to the lack of appropriate methods for extracting useful information from these complex, multidimensional datasets. Therefore, the uncertainty surrounding the clinical utility of the information and the lack of appropriate clinicians’ education represent major problems to be solved in the coming future. This review will summarize the technologies, advantages and limitations of two main genomic profiling approaches in oncological clinical practice, focusing on next-generation sequencing.
Comprehensive genomic testing for personalized treatments in advanced cancers
Molecular profiling uses a diverse methodology to find and characterize changes in the basic features of cancer cells. Next-generation sequencing (NGS), immunohistochemistry (IHC), fluorescent in situ hybridization (FISH) and quantitative polymerase chain reaction (qPCR) are examples of technologies used in molecular profiling. The traits analyzed in molecular profiling are called cancer biomarkers. Biomarkers may differ significantly, even among patients with the same type of cancer, and this is related to individual and diverse responses to the treatment. It is generally known that the effectiveness of patient treatment varies in cases where the same disease has been diagnosed and the same therapy has been used. Therefore, it is essential to characterize the tumor in detail. Cancer biomarkers are biological molecules, such as genes or proteins found in tissue or blood, that are part of a normal or abnormal process or are a determinant of disease. Individual biomarkers may have been changed in the process of malignant transformation, may be produced by the tumor itself, or may be the body’s specific response to the presence of cancer. Biomarkers can be informative about disease aggressiveness, identify the tendency to metastasize, provide clinically significant prognostic information, and indicate the presence or absence of drug-related characteristics.
One innovative approach combines next-generation sequencing with immunohistochemistry, providing a comprehensive view of the tumor profile, with information on DNA, mRNA and proteins, and allowing to identify further therapeutic options for the patient. This kind of test can also be performed on liquid biopsy specimens, either in combination with molecular tumor profiling, or alone. This approach is called theranostic solutions (a combination of diagnostics and therapy) and aims at providing cancer patients with the best treatment options.
Specific genetic alterations, unusual protein expression, or other biomarkers can be more effective for chemotherapies, targeted therapies, and immunotherapies decisions(18). This has been successfully demonstrated for a number of therapeutics targeting the protein products of specific genes that are altered in solid human cancers, such as Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2 or HER-2/neu) for trastuzumab, B-Raf Proto-Oncogene, Serine/Threonine Kinase (BRAF) for vemurafenib and dabrafenib, epidermal growth factor receptor (EGFR) for EGFR tyrosine kinase inhibitors or anti-EGFR antibodies, O-6-Methylguanine-DNA Methyltransferase (MGMT) promoter methylation status for temozolomide, the expression of programmed death (PD) ligand 1 (PD-L1) for anti-PD1 or anti-PD-L1 therapies in some solid tumors(19).
This comprehensive biomarker test is helpful in determining the best treatment(s) for patients with advanced solid tumors (e.g., breast, ovarian, stomach, colon, pancreatic, lung cancer, melanoma etc.). The analysis includes all biomarkers required to qualify a patient for approved biomarker-associated therapies – e.g., targeted and hormone therapies. Additionally, it gives an assessment of the prospective effectiveness of immunotherapy and a unique feature of this profile is the analysis of chemotherapy biomarkers. In the end, it identifies potential options for clinical trials in which the patient could participate. Such complex profiling is based on actual scientific reports and clinical data. The database and test content are regularly updated as new therapeutic options emerge, or new clinical data suggest the need to analyze new biomarker.
The theranostic approach is intended for patients with advanced solid tumors (stage III or IV) using Formalin-Fixed Paraffin-Embedded (FFPE) tissues. This type of test can be recommended upon diagnosis, when the first-line treatment is not efficient, in case of cancer recurrence, in highly aggressive or rare cancers, when the primary origin of cancer is unknown, or when treatment procedures have been exhausted.
Comprehensive molecular profiling shows significant value for rare, aggressive cancers and carcinomas of unknown primary origin (CUP), where treatment standards have not been defined. A critical asset of comprehensive tumor profiling is the unique IHC package differentiated for each type of cancer to identify the effectiveness of chemotherapies, specific targeted therapies and immunotherapy, consistent with the literature and clinical data.
A large number of genes (more than 600) are sequenced for SNS, Indels, CNV, introns for Alk/ROS1/RET translocation, MET-EX14, driver genes (RNA), gene fusions, and unusual splicing and genomic signatures, which include microsatellite instability (MSI) and tumor mutation burden (TMB), extremely important in assessing the effects of targeted therapies and immunotherapy. With the same profile, without the need for additional tests, the parameters of homologous recombination deficiency (HRD) are analyzed, predicting sensitivity to PARP inhibitors for homologous recombination deficient tumors. HRD is calculated based on algorithmic measurement of two factors: Loss of Heretezygosity (LOH) score, Telormeric Allelic Imbalance (TAI) score and BRCA1 and 2 splicings.
The combined solution of NGS and IHC together with the two technologies (Twist Biotechnology and Illumina Seq) ensures the sensitivity for SNVs/Indels to 5% and the CNV to six copies. The main sequencing coverage is 400X, with 99% on each target. MGMT promoter methylation is performed for glioblastoma, leiomyosarcoma, melanoma with brain metastases and CRC. Unusual splicings are analyzed for NSCLC (METex14), glioblastoma (EGRFvIII), and prostate cancer (AR-V7). The gene fusions are analyzed for ALK, RET, ROS1, FGFR1/2/3, and NTRK1/2/3 (cancer type dependent). The telomerase reverse transcriptase gene (TERT) is also included in the test.
In ten working days, the oncologists receive a dynamic report containing the most important information to determine new ways to customize patient’s treatment. These reports have a presentation of cancer with a picture of the tumor and a clinical form; a comprehensive list of variants and their biological impact; immunogram, showing the potential response to immunotherapy; a list of recommended versus not recommended treatments, a list of combination treatments with potential clinical benefit, potential lack of clinical benefit, treatment toxicity or undetermined clinical benefit, and a list of all current clinical trials. In the end, the list of all scientific publications is provided. Due to the uniqueness of each tumor, the oncologists must have a more accurate picture of the tumor in order to assess the effectiveness of various therapies as soon as possible. Studies have shown that NGS alone can identify the therapeutic options only for 27% of patients. The combination of the NGS technology with the unique determination of protein biomarkers (IHC) allows the identification of treatment options for as many as 92% of cancer patients(19). In this study 1057 advanced cancer patients from 30 countries (four continents) were profiled with comprehensive solutions after treatment(s) failure. Combining different techniques has led to better clinical insights. Ninety-two percent of oncologists who evaluated the reports agreed that the recommendations received after testing were useful. In a more recent study, 202 Chilean patients were sequenced by different tests commercially available. A significant percentage of patients (37%) could benefit from this approach, especially when NGS tests are combined with IHC(20).
Detecting molecular residual disease in solid tumors
After the cancer is treated, any cancer cells left in the body can become active and begin to multiply, causing the disease to come back. Their detection may indicate that the treatment was not completely effective or that the treatment was incomplete. Minimal residual disease, measurable residual disease, or molecular residual disease (MRD), refers to the small number of cancer cells that remain in the body after treatment. The number of remaining cells may be so small that they cause no physical signs or symptoms and often cannot even be detected by traditional methods such as imaging and/or looking for abnormal proteins in the blood serum. The minimal residual disease may be present after treatment because not all cancer cells have responded to therapy or because cancer cells have become resistant to the drugs used. Several tests can measure MRD. The more sensitive a test is, the more effective it is at finding a small number of cancer cells among many healthy cells.
Different methods can be used to quantify MRD: flow-cytometry, quantitative polymerase chain reaction (Q-PCR), digital PCR (ddPCR), and NGS(21). With the advance of sequencing technology and circulating tumor DNA (ctDNA) analysis, noninvasive monitoring has become feasible. Circulating tumor DNA has been proven effective in many solid cancers, such as lung cancer, breast cancer, colon cancer, or bladder cancer(22).
Since its discovery, in 1948, cell-free DNA (cfDNA) in human blood plasma has been extensively studied(23-25), demonstrating that ctDNA is an informative, specific and highly sensitive assay. MRD assay was used in a cohort of 55 early breast cancer patients undergoing neoadjuvant chemotherapy, following completing curative therapy to predict metastatic recurrence. Mutation monitoring in serial blood samples predicted recurrence with a median lead time of 7.9 months compared to demonstrated clinical recurrence(26).
In patients with stage I-III lung cancer, MRD was detected in posttreatment settings earlier than standard imaging, allowing oncologists to tailor adjuvant therapy to each patient at an early stage with the lowest disease burden(27).
In another study, MRD was analyzed before treatment in early-stage breast cancer and compared to clinical recurrence. The results showed that ctDNA detection had a median lead time of 10.7 months and was associated with recurrence in all breast cancer subtypes, suggesting that molecular relapse detection may be used to guide adjuvant treatment(28). In metastatic EGFR-mutant lung tumors, the detection of EGFR mutation in liquid biopsy after six weeks of therapy was linked to early progression on osimertinib with bevacizumab and to lower overall survival(29).
One of the most important aspects of liquid biopsy analysis is to fit into the rarity and heterogeneity of circulating tumor cells and the extremely low concentrations of circulating tumor DNA in the blood(30). Another issue is related to its ability to distinguish tumor-specific mutation from clonal hematopoiesis-related mutations. In this regard, the most accurate MRD detection method will include NGS sequencing from plasma and peripheral blood cells in parallel(31). Most of the available assays for detecting ctDNA from plasma are using fixed panels or looking for hotspots or actionable mutations. By this approach, even large panels might detect only a few mutations from a given individual’s primary tumor(32).
One innovative approach is to make MRD detection truly individualized and personalized for each patient using a personalized multiplex PCR (mPCR) next-generation sequencing assay (mPCR-NGS). According to authors, this is the first ctDNA assay that has been custom-built for detecting residual molecular disease and for assessing treatment response, with the ability to detect ctDNA at a variant allele frequency (VAF) <0.1% of cell-free DNA (cfDNA) from plasma, which is 10-fold lower compared to standard reported values of 0.1-1%(33-37). Somatic variants are identified by whole-exome sequencing (WES) of the primary tumor and the whole blood sample and, as a result, 16 clonal, somatic variants are generated for each patient. The resulting “tumor signature”, individualized to each patient’s tumor, is monitored throughout the patient’s disease course to detect the presence of tumor DNA in the plasma(34-37). A positive MRD test result means that residual (restorative) disease has been detected. A negative result means that residual disease was not detected.
Enabling ultra-deep sequencing without barcoding (100,000X average depth of coverage) on patient’s specific variants, this approach has a high level of confidence for a positive-ctDNA call, effectively going down to the single-molecule range. The limit of detection measured in variant allele frequency is 0.01%, which is translated in one mutant haploid genome in a background of 10,000 normal haploid genomes(34). The published analytical specificity is above 99.5%. The potential clinical usefulness of MRD assay, based on tumor signature, has been demonstrated in lung cancer, colorectal, breast, bladder cancers, and so forth(35-38).
Loupakis et al. analyzed a cohort of 112 patients with metastatic colorectal cancer (mCRC) who had undergone metastatic resection with curative intent. The study evaluated the prognostic value of ctDNA, by mPCR-NGS, correlating MRD status post-surgery with clinical outcomes(38). After surgery, 54.4% of patients were MRD positive, of which 96.7% showed disease recurrence associated with low overall survival (P=0.001)(38). In the MRD-negative arm, 96% of patients were alive, compared with 52.4% in the MRD-positive arm. MRD-negative patients who did not receive systemic therapy in the combined ctDNA analysis at two time points had an overall survival of 100%. The median time to progression for ctDNA-negative patients based on the first time point was 12.8 months versus 4 months in ctDNA-positive patients(38).
The clinical validity of ctDNA using mPCR-NGS was also assessed in patients treated by immune checkpoint blockade (ICB). A multicohort clinical trial of ICB in advanced solid tumors was conducted in a single-institution phase II study of pembrolizumab (INSPIRE)(39). Five parallel cohorts were included: squamous cell cancer of the head and neck (SCCHN), triple-negative breast cancer (TNBC), high-grade serous ovarian cancer (HGSOC), malignant melanoma, and mixed solid tumors (MST)(40). Early reduction in ctDNA after two cycles of pembrolizumab treatment and on-treatment ctDNA clearance was associated with a good prognosis, independent of tumor type, TMB or PD-L1 status. All 12 patients with ctDNA clearance during treatment were alive, with a median 25 months of follow-up(40).
Minimal residual disease testing can be recommended to evaluate how well cancer has responded to treatment; to confirm and monitor relapse; identify cancer recurrence earlier than other tests; identify patients who may be at higher risk of relapse; identify patients who may need to restart treatment; identify patients who may benefit from other treatments, such as stem cell transplantation or combination therapy.
The introduction of NGS sequencing in oncology has proven the applicability of biomarker-matching therapies in several cancer types. Common tumors as well as rare cancers can be targeted by specific therapies to improve clinical outcomes. Although biomarker testing shows promising results, there are a few limitations for their full implementation in routine practice. Some are related to the healthcare system, like poor access to targeted agents, cost of treatments, cost of testing and lack of clinical trial availability, while others are related to the testing itself, like the complexity of the molecular information generated, uncertainty regarding the clinical utility of the information, and clinicians’ education. We will list some of these, according to the feedback we obtained from Romanian oncologists. The main limitation expressed by oncologists is the cost of the test and, eventually, the treatment. Covering the cost of a test for the oncological patient is a problem that adds on top of other costs that come with the diagnosis itself. The recommendation from the medical oncologist for such testing is usually made only after other treatment options have been exhausted. In many patients, this overlaps with advanced cancers and poor prognostic. One way to improve this objection is by analyzing each case and identifying the patient who would benefit the most from such a test. Another limitation in the implementation of molecular profiling tests is related to possible off-label treatment recommendations that are difficult to support in the absence of adequate evidence and that can create anxiety for the patient. Likewise, the small number of clinical trials in which patients can receive therapy for free makes the oncologist’s work even more difficult. The complexity of the information that oncologists find in the report is also important for the oncologist’s daily practice since we still lack genomic standardization. Interpreting a report of this kind requires time and genomic knowledge that is not available to everyone. Considering the average/day of patients assigned to an oncologist and the complexity of the logistic documents that must be prepared, it becomes almost impossible for them to allocate the time necessary to understand, explain and evaluate the test result. As a result, many patients don’t have a complete understanding of the test benefits and often have unrealistic expectations from testing. Again, this issue can be improved if the oncologist will benefit from a free pre- and post-testing consultation from a specialized body. Pre-testing consultation (before testing) will give oncologists and patients sufficient information about the likelihood of finding a mutation in their specific tumor type for which a targeted treatment or clinical trial is available. Post-testing consultation will help oncologists in treatment decision-making by generating clarity on the report findings.
A significant part of the reluctance of doctors to use these tests is also related to the clinical usefulness of the information that the doctor will obtain. Especially if the test is reporting a low frequency of actionable alterations that are visible in many tumor types, the oncologist is facing the dilemma to recommend or not the therapy. To have a reliable interpretation of the molecular alterations in solid tumors, the test characteristics and the technical parameters regarding the quality together with tumor cell fraction calculation must be carefully evaluated. The decision to undergo genetic testing is based on an accurate understanding of the potential benefits and limitations of the test by the oncologist. There is also an urgent need, expressed by oncologists, to better understand these new diagnostic tools. This request is not surprising, given the content of current Romanian medical school genetics courses. Also, the low number of medical geneticists working in the medical healthcare system in Romania is holding back this process. Steeps have been made by including geneticists in the tumor molecular board or by genetic consultation in the oncological clinics where patients can seek additional information.
DNA sequencing has empowered oncologists and patients in their battle against cancer. Since molecular profiling has shortened the road from diagnosis to treatment, we expect a constant increase in the next-generation sequencing tumor panel offered to patients. However, many challenges must be overcome to implement these approaches in routine clinical practice efficiently. The constant effort of all parties involved in the healthcare system will make the promise of precision oncology a reality.
Acknowledgments. We thank the oncologists for sharing their opinions on molecular profiling tests.
Conflicts of interests: The authors declare no conflict of interests.