Technological advancements in cancer diagnostics: Improvements and limitations

Abstract Background Cancer is characterized by the rampant proliferation, growth, and infiltration of malignantly transformed cancer cells past their normal boundaries into adjacent tissues. It is the leading cause of death worldwide, responsible for approximately 19.3 million new diagnoses and 10 million deaths globally in 2020. In the United States alone, the estimated number of new diagnoses and deaths is 1.9 million and 609 360, respectively. Implementation of currently existing cancer diagnostic techniques such as positron emission tomography (PET), X‐ray computed tomography (CT), and magnetic resonance spectroscopy (MRS), and molecular diagnostic techniques, have enabled early detection rates and are instrumental not only for the therapeutic management of cancer patients, but also for early detection of the cancer itself. The effectiveness of these cancer screening programs are heavily dependent on the rate of accurate precursor lesion identification; an increased rate of identification allows for earlier onset treatment, thus decreasing the incidence of invasive cancer in the long‐term, and improving the overall prognosis. Although these diagnostic techniques are advantageous due to lack of invasiveness and easier accessibility within the clinical setting, several limitations such as optimal target definition, high signal to background ratio and associated artifacts hinder the accurate diagnosis of specific types of deep‐seated tumors, besides associated high cost. In this review we discuss various imaging, molecular, and low‐cost diagnostic tools and related technological advancements, to provide a better understanding of cancer diagnostics, unraveling new opportunities for effective management of cancer, particularly in low‐ and middle‐income countries (LMICs). Recent Findings Herein we discuss various technological advancements that are being utilized to construct an assortment of new diagnostic techniques that incorporate hardware, image reconstruction software, imaging devices, biomarkers, and even artificial intelligence algorithms, thereby providing a reliable diagnosis and analysis of the tumor. Also, we provide a brief account of alternative low cost‐effective cancer therapy devices (CryoPop®, LumaGEM®, MarginProbe®) and picture archiving and communication systems (PACS), emphasizing the need for multi‐disciplinary collaboration among radiologists, pathologists, and other involved specialties for improving cancer diagnostics. Conclusion Revolutionary technological advancements in cancer imaging and molecular biology techniques are indispensable for the accurate diagnosis and prognosis of cancer.


| INTRODUCTION
Increase in the rate of cancer incidence world-wide combined with enhanced mortality in some of the malignancies continues to pose a challenge to biomedical scientific community for an effective management of cancer. Prevention being a realistic probability only in few types of cancers, technological advancements in cancer diagnostics with precise determination of location, size, stage, and molecular characteristics, is urgently needed for cancer treatment, due to a worldwide increase in cancer related mortality. 1 Currently, the approach for diagnosis as a part of the clinical management of cancer includes a physical examination for abnormalities in various anatomical locations and a battery of laboratory investigations using blood and urine combined with a combination of radiologic and nuclear medicine based noninvasive imaging modalities like computerized X-ray scan (popularly referred to as CT scan), ultrasonography (US), magnetic resonance imaging (MRI), bone scan, positron emission tomography (using FDG, PSMA etc) followed by minimally invasive biopsy (needle aspirations) or invasive (surgical) biopsy coupled with histo-pathological examination to establish the identity and stage of the cancer. A number of immunological probes coupled with flow cytometric analysis are also widely used in the diagnosis and prognosis of liquid cancers namely, leukemias. While these approaches have been the backbone of diagnosis and treatment of cancers, they are nonspecific and also effective in moderately or highly advanced malignancies. Since early diagnosis of cancer has been found to improve the prognosis due to effectiveness of various therapies at this stage, and use of molecular targeted therapies significantly reduce the off-target effects (or side effects), there is a great deal of effort in developing diagnostic probes or biomarkers and approaches that target specific molecular and genetic abnormalities as well as highly sensitive analytical capabilities.
Cancer diagnosis is rapidly evolving due to continuous advancements in our knowledge of the disease and improvements in technology that increase the feasibility of reliable diagnostic approaches. 2,3 There are several cancer diagnostic modalities such as 2D and 3D imaging of tumors using positron emission tomography (PET), MRI, single photon emission computed tomography (SPECT), computed tomography (CT), X-ray imaging, and analysis of molecular (metabolic, proteomic, genomic, and transcriptomic) signatures of cancer cells, thereby leveraging the cancer diagnosis and management ( Figure 1). 4 However there is a lack of clinical effectiveness and cost effectiveness of various cancer diagnostic modalities, besides having strategies/ methods for evaluating associated risk and monitoring the therapeutic response. 5 Imaging is the most widely used tool to identify a diverse category of cancers based on various phenotypic properties associated with tissues within the tumor. 6 It is commonly used for the purposes of screening, staging, and monitoring tumor progression due to its accessibility and lack of invasiveness. 6 It is important to understand that the effectiveness of an imaging modality is heavily dependent on the growth rate of the solid tumor which can be represented by several different mathematical models: exponential, logistic, linear, surface, Mendelsohn, Gompertz, and Bertalanffy model. 7 The curve represented by the Gompertz model, for example, is a sigmoidal curve capturing the idea that a tumor's growth rate decreases as the mass of the tumor increases as a function of time. The reasoning behind this idea is that the proliferation of cancerous cells is highly dependent on the availability of factors such as nutrients and physical space, so as a tumor expands in size, the accessibility of these resources declines which ultimately leads to a slowed growth rate. Despite the existence of many tumor growth kinetic models, the Gompertz model has been shown to best represent solid tumors, primarily because it highlights a major characteristic of the vast majority of human cancers: they do not grow exponentially due to the doubling time, the number of days required for a tumor to double in volume, steadily increasing as the tumor grows rather than remaining constant. Modalities such as plain film X-ray, CT, US, MRI, and PET are the most commonly used to provide information about the physical structure, metabolic activity, and functional status of the cancer in the clinical scenario Table 1. 6 However, among each of these imaging modalities are inherent variations in resolution, sensitivity, and contrast generation which help to fulfill the primary principles and goal of cancer imaging: detection, characterization, and monitoring of tumors. 6,8 Detection refers to the localization of particular areas of interest within the image which allows for ability to characterize the tumor.
Characterization refers to the triad of determining the diagnosis, stage, and prognosis of the tumor. Finally, monitoring refers to the process of observing how the tumor progresses and impacts the rest of the body over time. The implementation and enhancement of these three main principles of cancer detection is what has allowed for a decrease in diagnostic ambiguity and inaccuracy which has led to improved patient care and outcomes as a whole. Moreover, methods of enhancing the process of generating an adequate signal to background ratio and threshold for detection still remains an area of investigation, besides reducing the artifacts associated with physical and physiological motions such as anatomical barriers and imaging time. 6,9 Additionally, the imaging methods like plain X-ray, CT, and PET uses ionizing radiation and radioactive material which warrants the need for advanced biodosimetry methods to prevent high dose radiation exposure during imaging. 10,11 By encompassing molecular diagnostic techniques, multi-parameter flow cytometry, immunohistochemistry, microarray, next generation sequencing, and other related molecular biology techniques, and nanomedicine, the breadth of diagnostics has grown significantly over the years. 6,[12][13][14][15] Despite the benefits of molecular diagnostic techniques in tumor classification, characterization, and precision medicine have been clinically demonstrated, inter-individual variations in the molecular signatures/pathways, validation methods, quality assurance, and high assay costs are some of the major limitations associated. [16][17][18][19][20] In this article, we will discuss various imaging and molecular diagnostic techniques commonly used for the detection of cancer, as well as rationales behind their actions, efficacies, and advancements.

| Positron emission tomography
Tomographic images produced by X-ray absorbance, magnetic resonance properties, and ultrasound reflection are imaging techniques that function based on the structural properties of the tumor. 6 Perhaps the most widely used imaging method present for the diagnosis of cancers is the PET, which is based on the functional status of the tumor tissue. A PET scan creates an image similar to a camera, but rather than creating an image by using visible light, it captures the simultaneous gamma rays generated by the annihilation of two positrons from a pharmaceutical agent that is localized differentially in the tumor tissue linked to its functional status to create an image. 21,22 First, certain isotopes of oxygen, nitrogen, and carbon generated in a cyclotron (a device that accelerates the particles) replace a hydrogen atom in a molecule of interest that emits a positron. A collision of a positron and electron occurs within the tissue, releasing gamma rays which are then detected by the PET scan. 21 The complex mechanism utilized by this diagnostic tool provides the foundation, not only for the three principles of cancer detection, but also for the clinical decision-making process regarding management according to a prospective cohort study executed by the National Oncologic PET Registry (NOPR). 23 This study collected data on the cancer management plan prior to and following analyzing the findings indicated on the PET scan via questionnaires. The findings of this study concluded that the post-PET management plan changed to monitoring and observing in 37% and treatment in 48% of the patients. In addition, about 70% of patients who were initially planning to undergo a biopsy were advised against that initial advice following completion of the PET scan.
Finally, in patients whose management plan consisted of treatment prior to and following the PET scan, the post-PET management strategy involved a significant change concerning the treatment type in 8.7% and treatment goals in 5.6%. Overall, physicians altered their initial management plan in 36.5% of cases due to the findings presented in the PET scan which exemplifies the great benefit provided by this imaging technique in oncologic settings.
Various types of PET imaging have been developed since its discovery. One of the most widely used pharmaceuticals is the positron F I G U R E 1 Technological advancements in cancer diagnostics labeled 2-fluoro-2-deoxy-D-glucose (FDG). 21 The image created by the PET scan is based upon the Warburg effect which states that high metabolic activity within cancer cells is due to increased glucose utilization in order to sustain continuous cell growth and division ( Figure 2). 24 Due to this phenotypic property, the positron emitting 18 fluorine ( 18 F) linked to the antimetabolite glucose analog 2-deoxy- However, interpretation of the results from a FDG-PET scan is made with caution as extraneous tissues like the brain, liver, and dense breast tissue may also have high FDG uptake and possess certain intrinsic characteristics that obscure the image. For example, brown fat serves the purpose of producing heat via its numerous mitochondria; the increased metabolic activity occurring within this tissue could induce FDG uptake and highlight areas that are actually cytologically normal. 26 New tracers are being developed that could potentially have greater sensitivity and specificity than the existing tracers. 2,7 One area of growth is individualized scans using tracers that are tailored to patients through gene profiling of their tumors. 21 Using specific tracers based on the circumstances of the patient may decrease background signal on the image and thus, create a higher quality image without any obscuration; this provides patients with the best diagnostic accuracy in the context of their clinical situation. 9,21 Specific radiotracers such as 11 C-and 18 F-choline, 11 C-methionine, 18 F-DOPA, 68 Ga-DOTA-somatostatin analogs, 68 Ga-ligand-prostate specific membrane antigen (PSMA), 18  that 18F-FET PET/CT added diagnostic information in brain stem and spinal cord glioma. 18F-FACBC has been shown to be a better PET imaging agent for prostrate tumor due to its slow metabolism that prevents its rapid accumulation in the urinary bladder. 40 Thymidine analogs such as 11C-thymidine, 3 0 -deoxy-3-18fluorothymidine (18F-FLT), and 1- Mechanism underlying the translocator protein (TSPO) binding used to measure the proliferation of tumor cells, targeting the thymidine uptake during DNA synthesis, suggesting the prognosis and aggressiveness of a tumor. 39,41 18F-labeled nitroimidazoles and Cu-labeled diacetylbis (N4-methylthiosemicarbazone) analogs PET-imaging has been utilized to measure hypoxia, implicating in determining the resistance of tumor to radio-chemo-therapy. 42 Therapeutic resistance has also been evaluated using various PET-based imaging biomarkers including sex hormone receptors, oncogenic receptors, and angiogenic factors. 39 Further, PETapoptosis imaging using 18F-ML-10 from the Aposense and 18F-CP18 (radiolabelled caspase 3 substrate) evaluates the extent of apoptosisinduced cell membrane asymmetry and acidification for assessing the therapeutic response in cancer patients. 43  and reproducible large data-set. 45 Involvement of multi-center and multi-disciplinary studies will over some of the limitations besides generating cross-validation algorithms and predictive models.

| X-ray computed tomography
Computed tomography is another method of imaging that can be utilized to diagnose cancer. CT has been effectively used for the screening of colon, lung, head and neck, breast cancers and so forth. with an accurate spatial and temporal tumor imaging, aiding in follow-up biopsy procedures, surgery, and radio-chemo-therapy. 46,47 Several instrumentation advancements such as scan speed, dual energy, iterative reconstruction, low kilovolt, perfusion imaging, and radiation dose reduction have accentuated the clinical utility of CT-based tumor imaging. 48 The spiral multi-detector CT with multi-fan measurement technique has resulted in improved spatial resolution, and has eliminated artifacts via high-end reconstruction and noise reduction algorithms. Photon counting (counting incoming photons and measuring their energy) and artificial intelligence has also enabled a high resolution CT image reconstruction, reduction in radiation dose, and artifacts. The CT scan is frequently used in addition to the PET scan to provide precise anatomical localization of the lesions revealed by PET. 24,49 The PET scan relies on the biochemical reactions taking place within the cells to form an image with low spatial resolution, while the CT scan creates an image with high spatial resolution that delineates the structural and morphological characteristics of the tumor. 24 Moreover, the concurrent CT scan corrects for the inherent attenuation seen with PET scans, helping to increase both the sensitivity and specificity of the image. 49

| Magnetic resonance spectroscopy
Magnetic resonance spectroscopy is a widely used form of imaging used for the diagnosis of cancer. It is most widely used in the diagnosis of brain tumors, but has more recently been applied in the diagnosis of pancreatic, prostate, breast, cervical, and gastrointestinal cancers. MRS differs from the conventional MRI in that signals from compounds such as carbon, hydrogen, creatinine, lactate, and N-acetylaspartate are measured rather than signals from water. 26,52 MRS is based on the concept of Larmor frequencies which demonstrates that protons in different compounds move at different frequencies based on the distribution of surrounding electrons. When a magnetic force is applied externally, the electrons generate a magnetic field since they are charged particles and proportionally shifts the frequency of the molecule. These changes in frequency are measured which can provide data regarding the composition of the area being imaged. 53 Rather than providing images of soft tissues, MRS quantifies various compounds like lactate, phosphocreatine (PCr), nucleotide triphosphate (NTP), phosphate monoesters, and inorganic phosphates; it also functions to determine the presence and varying amounts of these compounds in different target tissues which can potentially signify an abnormality. 54 The presence of lactate at the long time of echo (TE) when analyzing the brain indicates a pathological irregularity, suggesting the presence of a malignant lesion. 52

| Synthetic biomarkers
Synthetic biomarkers are a novel class of cancer diagnostic tool that uses a biosensor sensor placed inside the body to identify phenotypic changes at an early stage of the tumor and amplify this cancer related signals to a very high level that can be easily quantified. This approach is developed based on significant advances made in the areas of chemistry, synthetic biology and cell engineering and is for more sensitive than methods that analyze biomarkers that shed into the body fluids. 46

| Exosomes
Exosomes are extracellular vesicles secreted from many body cells that carry metabolites, RNAs (mRNA, miRNA, long non coding RNA), DNAs (mtDNA, ssDNA, dsDNA) and lipids from the cells in which they were generated and contribute to the intercellular communication. 47 Due to their stable nature, they are easily accessible as they are found in the bodily fluids like urine, plasma, saliva, and breast milk and bear a relationship with the cells of origin. They have been exploited as a biomarker for the early detection of cancer as their contents reflect the genotypic and/or phenotypic (aberrant proteins) alterations of the cancer cells they originated. 62 Due to their minimally invasive nature of access, they have an edge over the highly surgical tissue biopsy. 63

| Nano technology
Due to their small size, biosafety, better loading of diagnostic probe, and physical properties nanoparticles have been gainfully employed in various imaging based cancer diagnostics. Quantum dots that emit fluorescence in the near infrared region and have better tissue penetration has been used in combination with tumor specific biological probes (peptides, antibodies and other small molecules) for improved imaging, while silver-rich Ag2Te quantum dots that provides a better spatial resolution has also been used for tumor imaging. 64,65 Similarly, gold nano particles a good contrast agent with better biocompatibility and nanoshells have also been used for imaging of tumor tissue for early detection of malignancy. 66 Nanotechnology has also been used to assess the tumor microenvironment be exploiting the typical response of fluorescent nanoprobes to pH that helps in the detection of fibroblast activated protein-α in the tumorassociated fibroblasts. 67 The recent development of MXene based biosensors with high conductivity and superior fluorescent, optical, and plasmonic properties have been found be promising for the detection of cancer biomarkers due to their high sensitivity (femtomolar range for detection). 26 3.6 | Fluorescence in situ hybridization (FISH) tumor is composed of these cells. 85 The most imperative application of IHC in oncology today is specifically for the diagnosis of small round blue cell tumors and lymphomas as they identify the presence of specific "cluster of differentiation" (CD) markers which are used to identify specific cancers within these categories. 85 IHC is also used as a prognostic indicator for specific cancers such as breast cancer by identifying the presence of HER2/neu protein, estrogen and progesterone receptors, and markers of proliferation such as Ki 67. As IHC becomes a more prominent method of cancer diagnosis, its use is expanding for the detection of micrometastasis to lymph nodes. 85 Molecular methods of cancer diagnosis are gaining traction in the field of oncology. One of the most significant techniques in this category is the microarray which allows for the study of DNA, RNA, and proteins. 85,86 In the DNA microarray-based technique for the analysis of global gene expression, RNA is obtained and reverse transcribed using fluorescently labeled nucleotides resulting in labeled cDNA. 85,87 Hybridization of the cDNA with the preexisting probes in the microarray followed by scanning and analysis, provides important molecular information which can have significant diagnostic and prognostic implications. 85 For example, cDNA hybridization has been used to obtain prognostic information in patients with neuroblastoma. A cDNA microarray consisting of 5340 genes was obtained from primary neuroblastomas. These genes that were incorporated into the microarray were carefully selected as these specific sequences code for a protein that had an effect on the severity and prognosis of the malignancy. By utilizing this cDNA microarray to identify what genes were present, they were able to attain a prognostic outcome prediction that was accurate 88.5% of the time in the subjects that were studied. The microarray is advantageous in that it provides very specific objective information regarding a tumor. 87,88 It has also proven to be useful in patients with for precise interpretation of high-throughput data sets.
As we continue to study various cancers, begin to identify new tumor markers, and expand the reference database, molecular techniques such as the microarray and NGS will become more prominent methods used for cancer diagnosis (Figure 4). income status and those that live in low-and middle-income countries (LMICs) with resource-poor settings, especially due to high expense and lack of substantial medical infrastructure. 96 A great number of individuals globally live in resource-limited areas and therefore, have limited access to traditional diagnostic tools. This is a major concern, especially because an increase in cancer rates seems to be affecting developing countries disproportionately; cancer diagnosis are occurring at later stages with an increase in morbidity and mortality rates, and also are associated with a more expensive treatment plan. [96][97][98] This discrepancy has led to an interest in developing more low-cost diagnostic tools for a large fraction of the global population with minimal resources and underdeveloped healthcare settings.
For example, cervical cancer is the third most prevalent cancer affecting women globally, with a majority of cervical cancer deaths occurring in LMICs. 96,99 When targeting the issue of cervical cancer prevention, it is important to understand that there is an identifiable precancerous stage, also known as cervical dysplasia, which develops from oncogenic strains of human papilloma virus (HPV). This provides a longer period of time to recognize these precancerous squamous intraepithelial lesions to potentially prevent the progression to invasive stages. In 2018, there were approximately 569 000 new cases of F I G U R E 4 Next-generation sequencing and microarray as advanced tools for cancer diagnostics and personalized medicine cervical cancer and 311 000 deaths worldwide; around 84% of the new cases and 87% to 90% of the deaths occurred in LMICs. 100 Due to the high prevalence especially in LMICs, the World Health Organization (WHO) launched a global strategy in the attempt to eliminate cervical cancer as a major public health problem. 101   Thus, there is a need for more cost-effective and efficient devices to be implemented within the clinical setting so that physicians can screen for and treat various cancers in a timely manner.
In order to efficiently, accurately, and affordably diagnose diseases, it is essential that we thoroughly understand the current challenges regarding diagnostic tools and start placing more emphasis on the importance of using low-cost diagnostic and treatment technologies.

| Improvements needed in cancer diagnostics
Cancer diagnostic techniques are constantly undergoing developments and changes in order to successfully fulfill the primary goal of this field of medical care which is to provide accurate diagnosis in a timely manner. However, the presence of ongoing challenges within this field allows for the existence of opportunities to improve certain aspects of cancer diagnostics to quickly identify tumors, and accurately monitor tumor growth and metastasis. The first aspect of cancer diagnostics that can be improved is through the multi-disciplinary collaboration among radiologists, pathologists, and other involved specialties. Most often, the initial step in the cancer diagnostic process is imaging followed by a tissue biopsy; due to this two-step process, constant communication between radiologists and pathologists is necessary to ensure that the results of the biopsy performed directly correlate with the images gathered. 106 Having multi-disciplinary team discussions are crucial for making a definitive diagnosis and rational treatment plan via the combined knowledge and perspectives of the group, and for decreasing the amount of time associated with performing these diagnostic practices. 38 Another area of cancer diagnostics that could be refined to be more optimally efficient are the systems integrated within the various imaging modalities such as the picture archiving and communication system (PACS). 107 PACS is a computer network system used for the electronic storage and display of radiologic images rather than manually storing X-ray films. It is beneficial because it provides convenient storage and access to images from several imaging modalities, replaces conventional films with digital images, and allows the viewing of multiple images at the same time which is not something that can be done with conventional films. 107 After the imaging is completed, each image must be analyzed in multiple planes and this tends to be a very time-consuming process; thus, the more improvements we continue to make in the PACS system and in its interaction with other programs, the more we can facilitate the diagnostic process. 107 Because PACS is such a useful system, if we can continue to make slight modifications to make it more efficient but economical, we can potentially store radiology reports in a digitally-organized manner for easy access, better visualize and interpret images since they can be maneuvered through rotating and enlarging, and decrease cost by preventing the need to print films. New ideas, innovations, and enhancements should always be considered when trying to refine current technologies in cancer diagnostic techniques like the PACS system in order to achieve more reliable radiology tools and workflows.
Moreover, due to the ability to deliver multiple ligands and target receptors and other biological factors nanotechnology has proven to be an attractive approach in cancer diagnostics and therapeutics. Various types of nano-formulations like liposomes, iron oxide, dendrimers, quantum dots, gold nanoparticles, and carbon nanotubes are utilized for diagnostic application in optical, MRI, PET, CT, SPECT, and X-Rays techniques. 108 These nanoparticles can be targeted actively or passively targeted into the tumor for imaging and can be used as contrast agents (MRI and photoacoustic tomography). 111 In-DTPA-labeled pegylated liposomes have been used to image different types of cancers (breast brain, head and neck and lung cancer) using SPECT imaging. 108 18 F-liposomes is used in PET imaging and gadolinium-loaded nanoparticles in MRI imaging. Nanotechnology is advancing very rapidly and might prove critical in tumor imaging and therapeutics.
Through extensive interdisciplinary exchange of information between clinical and basic research will be vital in developing a potential futuristic roadmap, which will extend the realm of early cancer detection and diagnostics via developing technologies targeting vital cellular processes such as hypoxia, angiogenesis, and apoptosis, implicating in the management of refractory tumors. 109-111

| CONCLUSION
It is now well recognized that a proactive approach in the early diagnosis of cancer is key to enhance the efficacy of cancer management by improving the efficacy of most therapies with minimum side effects thereby providing extended survival with good quality of life.
Many conceptual and technological advancement that has taken place in the posthuman genomic area coupled with a blast in the computational technologies that the world has witnessed in the last few decades has not only strengthened the classical diagnostic methods that existed for more than several decades, but has also given rise to the development of a number of novel approaches for early, reliable and faster diagnosis of cancer. These include (but not limited to) Taken together the major practices utilized for the diagnosis of cancer, as well as their advantages, limitations, and areas for improvement. While some of these methods have been in use for a long time, new advancements in technology have their own clinical niche. As the field of medicine expands and we continue to strengthen our understanding of cancer, these diagnostic procedures will become more powerful and concomitantly, new techniques will be developed.

AUTHOR CONTRIBUTIONS
All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analy-