Including early surgical interventions will benefit in early precise detection and glioma molecular characteristics, early therapy decision-making and clinical trial interventions, shorter waiting times and longer survival times.
Glioblastomas are differentiated molecularly from other high-grade gliomas by the lack of an IDH-1 mutation (WT-IDH1 status), which increasingly dictates the poor prognosis and precision management of patients with this disease. Despite massive efforts and ongoing clinical trials, glioma patients have a median survival of approximately 14 to 15 months from the time of diagnosis. 3,22,23 Also, patients with high-grade gliomas who are transferred to rehabilitation centers experience longer lengths of stay, longer waiting times, and worse survival times than those who are discharged home. 26 Although there is a debate, based on the retrospectively reviewed and the large cohort study, supra-early chemo-radiotherapy after surgery (1–7 days) improves overall survival. 26,27
A proper diagnosis is the initial step in precision medicine. By providing samples for histopathology and molecular profiling, surgery plays a crucial role in the management of newly diagnosed and recurrent gliomas. 4,28 In order to diagnose and treat both newly diagnosed and recurring gliomas, a stereotactic biopsy or excision of the tumor is essential. In addition to molecular diagnosis, cytoreduction of the tumor to aid in optimal neo-adjuvant and adjuvant therapies and prolong overall survival are other indications for resective surgery. 4,28
Gliomas, particularly high-grade gliomas, are highly challenging to treat. Due to the heterogeneity of grade 2–4 gliomas, surgery is not a curative treatment, and adjuvant therapy is necessary to enhance the prognosis. 29 Resection of both enhancing and non-enhancing areas is advised for patients with Mutant-IDH1-glioma (WHO 3 astrocytoma or oligodendroglioma) and WHO 2-glioma based on the evidence-based advice for maximal extension of resection (EOR) in grade 2–4 gliomas. 4,28,29 Resection of merely the enhancing areas in elderly patients, or both the enhancing and non-enhancing regions, especially for younger patients, is advised in cases of WT-IDH1 status or glioblastoma. 4,29,49 On the other hand, glioma treatment is hampered by varying imaging accuracy. For instance, the enhancement of T1-weighted contrast in mutant-IDH1 WHO grade 3 astrocytomas and WHO grade 2 tumors is heterogeneous, and the removal of T2-enhanced tissue may result in microscopic pathology. 4,29 The T2-hyperintense areas' supramaximal resections remain controversial due to variable imaging accuracy. 4,30 It is imperative that future surgical and pharmacological clinical trials, treatment methods, and glioma classification techniques be improved. According to earlier research, specific surgical approaches for malignant astrocytomas may be considered based on IDH1 status since mutant-IDH1 requires supra-maximal resection surgery and its associated therapies that extend survival. 28 Therefore, creating a quick intraoperative assay is essential for increasing intraoperative assay accuracy and creating personalized surgical plans for mutant-IDH1 cancer. Knowing the mutant IDH1 status at the time of initial resection allows the surgeon to categorize the high-grade glioma and adjust the surgical approach to increase patient survival and lower the likelihood of repeat surgery. Additionally, further surgical and pharmacological clinical studies are needed for the treatment of gliomas, especially glioblastomas.
Early-IDH1 mutation detection with limited ethical concerns will improve Glioma management.
Currently, there is no CLIA-approved intraoperative assay for IDH1, which can guide the neurosurgeon and the extent of resection (EOR). Also, standard clinical assays such as the highly sensitive immuno-histochemistry, or high specific Sanger Sequencing or Next Generation Sequencing (NGS) methods that take on the order of days to weeks to deliver results. Although Immuno-histo-chemical staining (IHS) and sequencing are the most accurate approaches for identifying mutations, sequencing techniques like Sanger sequencing and Next Generation Sequencing (NGS), as same as imaging techniques, have lower sensitivity than the IHS method, which has low specificity. 31 Only one IDH1 mutation variation—the IDH1 R132H mutation—can be stained by IHS. 31 Furthermore, NGS or Sanger sequencing, brings bioethical concerns such as how to interpret and communicate the results, when to order, who to order for, and what kind of test to order must be taken into account.32 Also, sequence testing can raise ethical concerns owing to problems with consent, accessibility, results that are unclearly significant, unexpected and secondary findings, genetic discrimination, and the shared nature of genetic information. 32
IDH1 status is important for (Neo)adjuvant treatment in gliomas.
Chemo-radiotherapy can start right after partial gross tumor resection or biopsy surgery; thus, testing time and the time within surgery and adjuvant therapy can play a crucial role in glioma patient therapy. 27,33,34 Notably, Temozolomide (TMZ), irradiation, or their combination as standard (neo)adjuvant glioma therapy are more effective in treating mutant-IDH1 tumors. Mutant-IDH1 inhibitors have been put through clinical trials, but their effectiveness is constrained by their time-sensitive activity and the peculiarities of mutant-IDH1 passing alternation. 17,35 Also, mutant-IDH1 gliomas increase TMZ sensitivity and inhibit the ATM/CHK2 signaling pathway, which involves repairing DNA damage, in mutant-IDH1 gliomas that induce synthetic lethality. 36 Preclinical data and preliminary clinical activity from a previous study support the need for a preplanned subset analysis in mutant-IDH1 and WT-IDH1 gliomas in future clinical trials. 37 The expense and testing time could deter individuals from using the sequencing method. Even though there is more (neo)adjuvant therapy for gliomas, acute myeloid leukemia (AML), and several other cancer types is dependent on obtaining IDH1 status promptly. 17 Acquiring IDH1 status from the rapid and affordable assay with high specificity and sensitivity is beneficial for adjuvant Temozolomide efficacy and toxicity in glioma patients.
The Perioperative IDH1-LAMP assay can be used for calculating the individual therapeutic window of (neo)adjuvant therapy like Temozolomide as standard therapy, Metformin plus the antimalarial drug chloroquine or other trial medications, monitoring target therapy studies like IDH1 inhibitors, and immunotherapy studies by evaluating mutational load, amplifying mutant-IDH1 sequences, and replacing other techniques such as PCR. 38
Perioperative IDH1-LAMP assay with both colorimetric and fluorescent detection can detect IDH1 mutations accurately within 35 minutes of amplification.
Our efforts were focused on optimizing the LAMP and paper-based extraction methods to detect IDH1 mutations while suppressing WT-IDH1 variations in a rapid, reproducible, reliable, limited ethical concerns, and cost-effective fashion. In the experimental assay, the speed of the amplification is increased to only 35 minutes by modifying the concentrations of Betaine and LNA-primer mixtures and optimizing the Dipstick DNA extraction method. Additionally, utilizing virtual detection based on pH detection and fluorescent detection with qPCR equipment enables the identification of mutant-IDH1 according to the preferences of the test-taker and the patient's case. The Perioperative IDH1-LAMP can detect concentrations as low as 0.255 ng/ 25 µl or ratios as low as 1% of mutant-IDH1 in the background of WT-IDH1 via the fluorescent signaling method. This method allows for more accurate mutational load detection even in heterogeneous tumors where there may be areas of low mutant-IDH1: WT-IDH1 ratios, further optimized by colorimetric or fluorescent detection by a qPCR machine. Fluorescent detection by novel perioperative IDH1-LAMP provides the opportunity to detect mutant-IDH1 when tumor recognition by imaging approaches is difficult, allowing for assessment of super-maximal resection during surgery. Concluding IDH1 status via fluorescent signaling rather than colorimetric measurements has shown faster and less false-negative results. The assay can run in a constant water-bath temperature for 35 minutes if the IDH1 status is determined by colorimetric measurements. The presence of phenol red in the perioperative IDH1-LAMP assay and suppressing WT-IDH1 amplifications by IDH1-PNA, result in positive mutant-IDH1-R132 sequence amplification, increasing hydrogen ion byproducts and thus decreasing the pH of the solution, which leads to discoloration of the solution and fluorescent signaling detection while using qPCR due to present of the fluorescent mix in reaction solution (Supplementary material, Guide 5).
Our novel perioperative IDH1-LAMP assay demonstrates 100% specificity and 100% sensitivity when tested with positive and negative controls, even at low mutant-IDH1 DNA concentrations (0.1 ng/µl DNA in reaction mix). With improvements made in the assay and colorimetric detection, we can reliably detect the presence of the IDH1-R132 mutation not only in established cell lines but, more importantly, in fresh tumor samples obtained from the operating room in under 45 minutes. Our assay had 100% concordance of IDH1 status with our clinical standard Sanger sequencing and IHC testing.
The implications of rapid colorimetric or fluorescent IDH1-mutation detection can affect many aspects of and expedite patient care. The assay is developed to improve surgical super-maximal resection, increase quality of service process and service utilization, patient and provider satisfaction, encounters, develop an individualized therapy plan and personalized therapeutic window for (neo)adjuvant therapy, and facilitate in cost-effective medication alternation. With a short turn-around time, the IDH1 status can be reported intraoperatively to the neurosurgeon and oncology team while tumor resection is still ongoing. This can influence the extent of tumor resection, the aggressiveness of the surgery, particularly in the non-enhancing portions of the tumor, and the adjustment of (neo)adjuvant therapy. Early IDH1 characterization can potentially allow earlier inclusion into surgical and pharmaceutical clinical trials and make a larger range of the benefits mentioned available to the patient.
Supplementary material, Guide 4, is showing testing a tumor sample with control samples in stepwise fashion. To the best of our knowledge, the novel intraoperative IDH1-LAMP assay is the fastest test from sample collection from operation room, delivery to the laboratory, sample preparation, running the amplification, and identifying IDH1 status in 45 minutes with considering the affordability and accessibility of the materials. With this turnaround time, shorter surgical procedures and clinical judgments are possible, which can be cost-effective. The suggested pre-operative assays could be performed on DNA from epithelial cells that has been extracted and precipitated simply using just saline and 100% ethanol (Supplementary material, Guide 4), (data not shown), as an assay quality control, validating primers and IDH1-PNA sequences, and individualized the test before surgery as a single mismatch can affect IDH1-PNA efficacy and assay results significantly. 39,40 Also, the web data-based prediction tools like the Ensembl genome browser and NIH algorithms can be utilized to identify SNPs. 41 The Pre-operative assay has the potential to be used as a screening tool for identifying germline mutations and single nucleotide polymorphism (SNP) variations at codons 130–135 on IDH1 that may associate with a disease and as a marker for response to therapy.
Peri-Operative LAMP assay has the potential to be utilized for other cancer types or disease associated with IDH1 mutation.
Our intraoperative LAMP assay can be used to detect any cancer or condition that has an associated germline or somatic IDH1-R132 mutation. In small bowel adenocarcinomas (SBAs) linked to Crohn's disease, IDH1 gene mutations are more prevalent than in sporadic (non-inherited) cases, but APC mutations appear to be less common in the former. 42 Notably, IDH1 mutations are seen in gliomas and enchondromas, noncancerous (benign) cartilage growths that form inside the bones in Maffucci patients. 43,44,45 The first case of a pituitary adenoma with an IDH1 mutation has been reported. 46 Pituitary adenomas have traditionally been the most prevalent sellaturcica tumors, but malignant gliomas are one of the uncommon forms of pituitary tumors that may go unnoticed on a skull radiograph. 47 IDH1 mutations have been reported to associate with Glioma, AML, Ollier, and Maffucci syndromes. Based on the National Cancer Institute (NIH) GDC data portal, Mutant-IDH1-R132 has been reported in various primary resources, including the brain, hematopoietic and reticuloendothelial systems, prostate gland, bladder, breast, colon, liver, intrahepatic bile ducts, skin, bronchus and lung, connective and soft tissue, and thymus.
Future studies and Study limitations
Upregulated WT-IDH1 expression in glioma, imbalanced redox homeostasis by increasing the NADPH/NDP + and reduced/oxidized glutathione ratio, and independent IDH1 fatty acid synthase in glioma led to the identification of WT-IDH1 as a potential target therapy. 48 Precision medicine for glioblastoma or probably other cancers or diseases related to IDH1 functions via developed IDH1-PNA can be studied to inhibit DNA transcription, as demonstrated by data, and reduce tumor weight; Data are not shown. An in Vitro study showed an 80% reduction in tumor weight, but further investigations are needed to determine a therapeutic formulation and delivery strategy. Future trials and research are suggested in following, based on the literature review and the current study. The initial supra-maximal resection and individualized surgical method for mutant-IDH1 gliomas should be performed with intraoperative IDH1 mutation assay (1). Only mutant-IDH1 inhibitors or combination therapy, including chemo-radiotherapy with other trails medications should be used in conjunction with intraoperative IDH1 mutation confirmation if supra-maximal resection of mutant-IDH1-glioma is not possible (2). Using non-TMZ (Neo)adjuvant trials or IDH1-PNA with a therapeutic formulation or delivery mechanism right after surgically eradicating the enhancing portions of WT-IDH1 gliomas, especially in geriatric patients, or, if possible, both enhancing and non-enhancing regions, especially in younger patients (3). If KPS of glioblastoma patients is low, (Neo)adjuvant trials or IDH1-PNA with a therapeutic formulation or delivery method should be used in clinical trials (4). Reverse-transcriptase LAMP and mRNA sequences can be developed to potentially improve sensitivity, but RNA extraction, RNA and enzyme stability, and the labor-intensive process of making cDNA from RNA will be problematic. However, considering lyophilizing or freeze-drying reaction mixtures may increase speed and DNA yields in reaction mixture.
The limitations of this study include the small number of patient samples included in this study, with only one out of five harboring an IDH1 mutation. While our data shows extremely encouraging results, more rigor with a larger number of samples in both a single and multi-institutional setting is warranted.