July 20, 2024

The last ten years have seen a sharp increase in the application of immunohistochemistry (IHC) and immunofluorescence (IF), particularly in recent years. While research skills and technology have progressed in tandem with them, certain laboratories may find themselves faced with an abundance of options. Joe Poh sheng Yeong, a research immunopathologist at Singapore General Hospital, argues that newcomers to the area might feel torn between enthusiasm and confusion about a bright future. But multiplex IF offers a clear path to that promising future.

Read More: spatial phenotyping

According to Matt Humphries, scientific lead for tissue hybridization and digital pathology at Queen’s University Belfast, multiplex IF opens the door to spatial phenotyping, which allows labs to “not only identify several cell types within a single sample, but also categorize whether a particular cell is expressing several biomarkers – allowing us to recognize specific phenotypes.” “This high level of cell profiling could address treatment regimen failures in clinical trials, particularly in immuno-oncology, and hold significant prognostic information at a basic diagnostic level.”

An unfulfilled need

The response rates to current immunotherapy are dismal, with less than 20% of cancer types responding. As a community of immuno-oncology researchers, we aim for tailored medication, which might enhance this, adds Yeong. “But, areas with limited resources continue to face issues with accessibility and technology, as well as long turnaround times and financial constraints.”

Not only that, but previous to the emergence of spatial phenotyping, transcriptome analysis and next-generation sequencing (NGS) were the primary methods used to predict treatment response. “The loss of spatial arrangement of influential cell types is a significant drawback, even though these techniques are essential for gaining high quality subvisual data,” notes Humphries. “Even with phenotypes that are expressed at very low levels, maintaining the morphological landscape can help us identify key phenotypes that can impact tumor progression or patient survival.”

Difficulties with big data

Another difficulty in biomarker research is the creation of big data. It is anticipated that clinicians would examine hundreds to thousands of samples, with individual whole-slide multiplex IF pictures occasionally reaching sizes of up to 100 GB. Humphries states, “Especially within the timeframe that diagnosticians have to assess a single slide, it is unrealistic to expect the human eye to assess and quantify an image containing hundreds of thousands of data points and report these with a high degree of accuracy and reproducibility.”

Simplifying the massive data flow that will help researchers find biomarkers for cancer is becoming more and more important. Humphries notes that computational pathology can quickly and accurately measure the amount of cells expressing two biomarkers within a certain proximity to tumor cells, a task that would take a doctor many hours to perform. However, who bears the duty for advancing it? “An oncologist choosing a patient’s treatment plan may find this high-level information extremely helpful, but laboratory medicine professionals will need to authorize such analysis.”

certain benefits

For patients to receive the best care possible, it is essential to predict the outcome of immunotherapy. While there are several approaches available, spatial phenotyping has the benefit of requiring fewer tissue slides. Humphries emphasizes that “human tissue is precious and can be a finite resource if predicting immunotherapy response requires the assessment of several biomarkers, whether used for research or clinical diagnosis.”

“Not only does this require multiple slides for individual analysis (which could be limited if the tissue is a small diagnostic biopsy or a valuable tissue microarray research resource), but the topography of the tissue changes as you move through a specimen because each subsequent slide is stained and reviewed,” he continues.

How may this be resolved using multiplex IF? According to Humphries, “spatial phenotyping can capture all these biomarkers and their cellular landscape in one slide.” Additionally, cell types that co-express biomarkers with possible prognostic significance can be identified using multiplex IF. Spatial analysis is useful when these characteristics are close together since the volume of data there may be a significant indicator of an immunotherapy response.

Yeong concurs, saying “the advantages are tremendous.” Nobody involved with cancer immunotherapy could likely contest that. When examining phenotypes that necessitate identifying multiple markers, we can preserve the tissue for examining two markers on a single slide. This allows us to conduct our investigations in a manner that is compatible with the majority of digital pathology analytical software for thorough interpretation, including high-dimensional and spatial analysis. Additionally, it is compatible with and has a lot of promise for clinical translation.

taking the lead

Yeong thinks early adopters of spatial phenotyping with multiplex IF have a lot to look forward to in terms of what lies ahead. Similar to those that embraced molecular testing and NGS all those years ago, labs won’t have to outsource to a third-party lab. Rather, their internal researchers will possess the expertise to comprehend and analyze the information. However, he adds, “Oncologists and surgeons will no longer have to worry about this part of immunopathological monitoring if the lab is a part of an Academic Medical Center or National Cancer Center. These days, it’s like having an indispensable arm in most large trials and studies.”

According to Humphries, “Laboratories using multiplex IF for spatial phenotyping will quickly realize the enormous wealth of information contained within a humble tissue slide.” In order to meaningfully affect patient survival, there will be an increasing need for precise, objective analytical goals as the potential for more nuanced data extraction develops.

Avoid falling behind.

Research labs that do not use spatial phenotyping run the danger of having their expansion stifled. Humphries states, “They will continue to provide the excellent diagnosis and reporting that they are currently capable of, and only that.” “The laboratories that push the envelope with these new methods will truly reap the benefits, and there will be a point of critical mass when industry, national health agencies, and patient needs will drive adoption,” he says, acknowledging that caution is understandable when new technology challenges the status quo. The advent of methods and tools like IHC, high-throughput auto-staining systems, and digital pathology has already demonstrated this in recent medical history.

But, there are organizations devoted to assisting laboratories in adapting, so it’s not all bad news for those who haven’t made the switch yet. “I am a member of the JEDI council, a task group. Yeong states, “Our objective is to increase accessibility to the knowledge of staining, imaging, troubleshooting, analysis, interpretation, and reporting standards and quality control.” “And there are more—many other international committees and task forces are already contributing to this endeavor.”

Additionally, laboratories might begin small when pursuing spatial phenotyping. In order to validate our findings in single-plex analysis, Humphries adds, “we started tentatively with small panels. One of these was in an esophageal adenocarcinoma cohort demonstrating a dual-positive phenotype that could have prognostic value.” But our trust in panel design and application, as well as our in-house technical ability, have evolved along with our panels.

Leaving knowledge behind

Yeong and Humphries have an important message for pathologists and laboratory medicine practitioners everywhere, regardless of where they are in their spatial phenotyping journey. Yeong states, “There is still more to come. The field of oncology has evolved from the ‘H&E-only’ era to IHC, molecular testing, and now cancer immunotherapy.” “In order to employ multiplex IHC and IF to advance the field and get past financial constraints and sluggish turnaround times, we need teamwork and shared wisdom.”

Humphries concurs that the time has come for labs to implement spatial phenotyping and lead by example. Remember to start having discussions about new technology as soon as you can. Early acceptance of new technology would take significantly longer in the absence of your knowledgeable and distinct perspective, he argues. My mission as a translational scientist is to assist pathologists and laboratory medicine specialists in their outstanding work. If innovative methods of operation may enhance specialized clinical expertise, reduce workload, and enhance patient outcomes, I hope this is a route that all scientists would wish to take.