Pathology Ushers in a New Era of Cancer Prevention

2023/04/27

Professor Yae Kanai says she knew she would spend the rest of her life dedicated to pathology when she took her first pathology course as a medical student at Keio University. After three years of graduate school, she submitted her dissertation and went on to the National Cancer Center Research Institute, where the young pathologist made a breakthrough in global cancer research when she published one of the world's earliest papers detailing how abnormal DNA methylation can occur at precancerous stages. Prof. Kanai returned to her alma mater some twenty-five years later, in 2015, and is now breaking new ground to apply her research findings to the early diagnosis and prevention of cancer.

From an early age, Prof. Kanai had aspirations to become a doctor. However, as a medical student, her initial desire to become a clinician transformed into a desire to do research that could contribute to better clinical practice. “I wanted to pursue a career unique to me and my skill set, doing a job where I could create something new. But life is long, so I felt anxious about the thought of conducting research for decades to come.”

Prof. Kanai decided to pursue a career in pathology, believing that if she could base it around morphology, she would build a lasting foundation for a life of research.

“Pathology is the scientific study of disease and is rooted in morphology, which is the observation of human tissue under a microscope. We use our eyes to see and aggregate visual information to understand the tissue. That’s why when training to be a pathologist, it’s important to be hands-on—looking through the microscope and observing tissue in detail with your own eyes.”

Prof. Kanai says that as a student, she enjoyed using microscopes and looking at histology textbooks with their collections of micrographs.

“Each type of tissue has a different form and function. It is never random and is always arranged in an orderly fashion. Before I knew it, I was completely taken by the beauty of human tissue under the microscope. Since function can only be endowed in morphological structure, I figured morphology would be a strong foundation to keep me grounded even if my research continued for decades to come.”

And just as Prof. Kanai had suspected at the tender age of 20, pathology has become a lifelong pursuit.

Students who go on to study pathology at graduate school typically do research and specialist training concurrently.

“A pathologist's work, based on microscopic observation, has two sides. One side is conducting studies as a researcher, and the other is diagnosing patient samples as a clinical pathologist. As both a clinician and a researcher, you have to do twice the work because you have to be on both sides.”

But Prof. Kanai believes that aspect to be the real thrill of pathology—the ability to conduct research and clinical work simultaneously.

“Pathologists are in the difficult position of straddling the line between the hospital and the laboratory, yet we believe it is ideal to have one foot in each world. Rather than looking into the microscope as a clinician whose sole purpose is to treat patients, I think the mark of a true pathologist is their ability to conduct research with the mindset of a clinician and to diagnose patients with the scientific eye of a researcher. In short, we can make diagnoses based on research or conduct research based on diagnoses. By doing both simultaneously, I believe that we can create something greater than the sum of its parts, and this idea also resonates with the graduate students who join my research lab. When you first go into pathology, it’s challenging just to perform autopsies on your own, and the pathologist exam is so tough that even practicing pathologists sometimes fail the exam. So I understand that graduate students studying pathology can have a particularly hard time, but I am also pleased to see that we continue to attract students who want to pursue both research and clinical work.”

During her graduate school days, Prof. Kanai did not simply follow convention but acted as a pioneer.

“I studied under a professor who specialized in the pathology of autoimmune diseases and was tasked with assignments such as purely morphological analysis, but I also used my own judgment and did plenty of things that no graduate student had done before. Things like remodeling a warehouse to build a new laboratory and starting joint research projects with researchers from other departments. To broaden my horizons and expertise, I finished my dissertation in three years and moved to the National Cancer Center Research Institute while still a graduate student. There I was able to develop molecular pathology that pursued the molecular biology of what I saw under the microscope.”

It was at that time that molecular biological analysis of patient tissue samples was becoming possible for the first time.

“With the Pathology Division at the National Cancer Center Research Institute receiving a daily stream of pathological diagnoses, we were able to extract nucleic acids from samples of consenting patients, which positioned us to become one of the first laboratories in Japan to harness these new molecular biology techniques. At that time, I was fully immersed in my research on cell adhesion.”

In normal tissue, neighboring cells firmly connect themselves to each other, as if holding hands, in a process known as “cell adhesion.”

“Cell adhesion plays an important role in creating the beautiful, art-like tissue architecture we see under the microscope. If a cell is ‘holding hands’ with a neighboring cell, it is unable to act on its own. The clean alignment of cells in a certain direction is called polarity, and if polarity is properly maintained, multiple cells can work together to fulfill their functions. And cell adhesion is critical in maintaining that polarity.”

If they let go (i.e., a breakdown between cell adhesion molecules), the cells will break away from each other. This means that the beautiful network that cells construct with each other is disrupted, which is linked to the formation of cancer and the invasion of metastatic cancer cells. Cell adhesion molecules, which serve as those hand-holding molecules between cells, proved very important to Prof. Kanai’s research not only because they shape the morphology of cells but also because they are involved in the very nature of cancer development and progression.

“At the time, the human genome (all the genetic information in DNA) had yet to be decoded, and the base sequences of cell adhesion molecules themselves were not yet understood. Since the normal human nucleotide sequence was not yet known, we had to determine the full-length nucleotide sequences of genes encoding cell adhesion molecules and register them in a database before examining cancer cells for abnormalities.”

Most cancers arise from epithelial cells that cover the surfaces of the body and body cavity, such as the lumen of the gastrointestinal tract. Epithelial cells have developed a potent cell adhesion apparatus called the cadherin-catenin system. In studying the abnormalities of multiple cell adhesion molecules that comprise the cadherin-catenin system in various types of cancers, Prof. Kanai discovered that the expression of E-cadherin, one of the most powerful cell adhesion molecules, can be silenced by a mechanism known as DNA methylation alteration.

“DNA methylation is one of the alterations referred to as ‘epigenetic modification.’ When I first focused on DNA methylation ahead of the global research trend, these modifications were treated as superficial mutations and were not thought to play a major role in carcinogenesis, so they were not considered to be of much importance. At the time, the field was dominated by the idea that cancer was caused by genetic mutations. Very few people were studying DNA methylation, so we needed to accumulate and build upon our research findings to overcome that skepticism.”

After gaining experience as part of a cell adhesion research team, Prof. Kanai decided it was time to embark on her own independent research in her mid-thirties, just as her team discovered that E-cadherin could be silenced by DNA methylation.

As a cell adhesion molecule, E-cadherin can function as a tumor suppressor gene due to its ability to prevent abnormal adhesion between cancer cells. When Prof. Kanai and her colleagues first reported that E-cadherin could be silenced by DNA methylation, it was considered highly exceptional for a tumor suppressor gene to be inactivated by DNA methylation alterations.

“It was so rare that people said it probably had little significance. The ‘two-hit theory,’ first proposed as the mechanism of tumor formation, states that for cancer to occur, both paired tumor suppressor genes must be inactivated at the same position on the chromosome. Inactivation was thought to occur only when two conditions were met: (1) a part of the genome was lost, and (2) a gene mutation occurred. However, our demonstration of E-cadherin inactivation by DNA methylation showed that it is a universal process and that DNA methylation inactivates tumor suppressor genes and fulfills the two-hit theory even without a gene mutation, as was previously thought.”

At the same time, Prof. Kanai was routinely observing precancerous conditions under her microscope and concluded it would be important to understand what is happening in the early stages of tumor formation to prevent and treat cancer.

“Since genetic mutations don’t occur at the early stages of multistage carcinogenesis, I wondered what mechanism they are preceded by. That’s when DNA methylation came to mind. Abnormal DNA methylation was also said to alter the chromatin structure, resulting in chromosomal instability, which was a logical explanation. Because we were observing precancerous stages through a microscope, we were able to observe in detail how DNA methylation can drastically change the tumor microenvironment and how cancer cells adhere and break apart in response to their environment. When cancer cells invade blood vessels and begin distant metastasis, they are broken up individually because they have to pass through 'enemies' such as blood and surrounding cells. However, when cancer invades a target organ to expand its power, it is difficult for cancer cells to do so on their own, so they end up asking their neighboring cancer cells for help and once more ‘join hands’ with nearby cells and adhere to them.

“Methylation is one of the epigenetic mechanisms and an example of how cells can regulate gene function without changing the DNA base sequence. Even when the world wasn’t paying any attention, I continued my research, believing that one day, epigenetics could be shown to play an important role in carcinogenesis. We had been studying liver cancer, and I think it was significant that we were able to directly prove—using human samples exhibiting precancerous conditions such as hepatitis C and cirrhosis—that abnormal DNA methylation already occurs at the same chromosomal sites where deletions would be observed in liver cancer themselves.”

In this way, Prof. Kanai became one of the first researchers in the world to report that abnormal DNA methylation occurs in the precancerous stage, thus significantly advancing cancer research. Today, it is commonly known that most tumor suppressor genes are inactivated by DNA methylation.

Prof. Kanai says that she considers this to be the start of her career as an independent researcher, and she and her colleagues are now taking their years of experience studying methylation and working to apply it in new ways, including innovative cancer risk diagnosis methods.

“Once DNA methylation profiles are established at the precancerous stage, such patterns tend to be maintained and are inherited by cancers themselves. And so, we continue to study the application of DNA methylation as a biomarker for various purposes, such as diagnosing cancer risk, detecting and preventing cancer, and predicting how patients will respond to cancer treatment.”

Today, one disease Prof. Kanai believes should be the focus of diagnosing cancer risk is non-alcoholic steatohepatitis (NASH).

“With NASH, fat accumulates in the liver over many years, and as inflammation continues, it leads to cirrhosis and eventually liver cancer. It is estimated that cirrhosis will progress to liver cancer in more than 10% of patients. Although NASH is on the rise as a metabolic disorder, many patients do not initially have symptoms and do not necessarily make regular visits to the hospital, even after being diagnosed. When confirming a NASH diagnosis, we perform a liver biopsy, the idea being to use a portion of the biopsy to measure whether the patient has the type of DNA methylation profile that would predispose them to cancer in the future. We believe that informing people of such risks will create opportunities for them to engage in treatment to stop NASH from progressing, and with clinicians also wanting access to this kind of data, we are continuing to develop tests for medical application.”

Prof. Kanai and her colleagues have been working to develop a method that is easy to implement and suitable for clinical specimens containing a mixture of different cell lineages. Now, through collaborations between industry and academia, they have succeeded in creating a diagnostic method using high-performance liquid chromatography (HPLC).

“We use samples left over from diagnostic liver biopsies, so there isn’t any extra burden on the patient. Using the HPLC method, we can obtain test results in about ten minutes. It is easy to implement the system at hospital laboratories, so we hope to promote a system where we can inform the patient on how best to prevent NASH, stop the disease from progressing, and, in doing so, avoid more people developing liver cancer.”

Upper urinary tract cancer is another disease Prof. Kanai hopes to apply DNA methylation diagnosis to as soon as possible.

“Upper urinary tract cancer is the same urothelial carcinoma as bladder cancer, but it develops in the renal pelvis and ureters, which are upstream of the bladder. It is known to be difficult to diagnose, and it can progress during the time it takes to do so. We are currently conducting a multi-center joint research project with the aim of diagnosing upper urinary tract cancer by urinalysis alone. At the National Cancer Center, I specialized in diagnosing urologic cancers, so I was always in contact with urologists over the phone or at conferences. We’ve always had a close relationship where they could come to me with a pathology report and ask me to show them their sample under the microscope. Those same urologists are now in leadership positions throughout the country, and together we have formed a urology consortium and are starting to conduct studies. The sensitivity and specificity we have achieved with DNA methylation diagnosis are excellent, and we think it will prove useful.”

Even as she makes strides with the clinical application of her DNA methylation research, Prof. Kanai's work continues to combine the fields of molecular biology and morphology to take her research in other new directions.

“It is now well understood that molecular information is big data, but morphological forms are also a kind of big data. Until now, the microscope has been the only means of managing data related to morphology. I guess you could say that specially trained pathologists have diagnosed cancer by managing the morphological ‘big data’ seen under the microscope. Looking under the microscope means looking at the totality of the vast number of genetic abnormalities behind morphological abnormalities. At the same time, the essence of examining samples under a microscope can also be seen in aspects of omics analysis, which is now widely used, for example, to synthesize and profile the DNA methylation status of a vast number of genes. I think that such morphological and molecular information could be integrated with AI using deep learning to create new genomic medicine. With such an experienced understanding of both pathological diagnosis and molecular pathological research, I believe that our lab is uniquely positioned to tackle the research that will lead to new breakthroughs in genomic medicine.”

Yae Kanai
Prof. Kanai graduated from the Keio University School of Medicine in 1989. In 1993, she completed a doctoral program in pathology at the Keio University Graduate School of Medicine and began working as a researcher in the Pathology Division at Japan’s National Cancer Center Research Institute. In 2002, she became director of the Pathology Division at the National Cancer Center Research Institute and, in 2010, went on to serve concurrent roles as the director of the Molecular Pathology Division and deputy director (until 2014) at the institute. Since 2015, Prof. Kanai has served as a professor in the Department of Pathology at the Keio University School of Medicine. Since 2021, she has served as a vice dean of the Keio University School of Medicine.