Epic journey yields unique offering
Eye on Innovation
Eye on Innovation features exciting advances taking place at Mayo Clinic Laboratories. This monthly series shines a spotlight on recently developed tests and highlights how Mayo Clinic translates ideas and discoveries into testing resources that improve diagnosis and care for patients across the globe.
Like any notable odyssey, the one undertaken at Mayo Clinic Laboratories to develop a test to detect a debilitating autoimmune neurological disease was a decades-spanning adventure that passed through the unknown and consumed the energies of the neurologists navigating the journey.
With steadfast direction from patient-focused leaders in the Neuroimmunology Laboratory, and fortuitous findings propelling the journey, the quest to create a test pinpointing the source of the rare, testicular cancer-associated illness successfully concluded in the summer of 2021.
The odyssey’s outcome was the world’s first test offering to detect antibodies to the Kelch-like protein 11 (KLHL11), a biomarker for testicular cancer-associated paraneoplastic encephalitis. Implemented in June, the inventive test uses a mechanized cell-based assay that employs pioneering slide-scanner technology to screen patients and then tissue immunofluorescence to confirm disease presence.
Divyanshu Dubey, M.B.B.S.,
“In the new paraneoplastic criteria, only a handful of antibodies are considered a high-risk cancer antibody, and Kelch-11 is one of them,” says Mayo Clinic neurologist and lab medicine physician Divyanshu Dubey, M.B.B.S., who became interested in the condition during his autoimmune neurology fellowship at Mayo Clinic. “It’s a crucial antibody with very strong cancer associations, so it can not only help with early diagnosis and to develop a treatment plan, but to find the cancer. So the test has multiple implications.”
Since implementation, the stand-alone test (ID: K11CC or K11CS) has been ordered more than a hundred times since going live, with increasing test requests each month. Among patients tested, 10 have been identified as positive for the antibodies. Estimated to occur in three per 100,000 men, the disease is one of the most common paraneoplastic neurological disorders in young or middle-aged men, Dr. Dubey says.
“The test truly highlights how people have recognized that this is a disease with a unique biomarker designation,” says Dr. Dubey. “It can be challenging in many of these cases to make a diagnosis because most of the time, patients present with neurological problems before they are able to detect the testicular cancer. Having a biomarker allows people who don’t have a lot of experience with paraneoplastic illness to guide them to the disease.”
Sean Pittock, M.D.,
While the world at large has only recently recognized testicular cancer-associated paraneoplastic encephalitis as a distinct disease state, Dr. Dubey and his neuroimmunology laboratory colleagues Sean Pittock, M.D., co-director of the Neuroimmunology Laboratory, and Vanda Lennon, M.D., Ph.D., former director and founder of the Neuroimmunology Laboratory, have long recognized the distinct nature of the illness.
In the late 1990s, Dr. Lennon first recognized the illness in a male patient suffering from walking difficulty. Over the next 15 years, Dr. Pittock noted additional cases. When Dr. Dubey joined the lab in 2017, he took over efforts for the autoantigen that caused strikingly similar symptoms in patients.
“As a fellow, I was seeing handfuls of cases in neuroimmunology clinical laboratory who had ‘sparkles’ pattern on tissue immunofluorescence assay (TIFA),” Dr. Dubey says. “And every time I reached out to the managing physician to ask what was going on with the patient, patients’ clinical syndromes were very similar.”
Vanda Lennon, M.D., Ph.D.,
The consistency of symptoms in patients whose specimens exuded the “sparkles” pattern was unwavering, Dr. Dubey says. “The patients had walking difficulty, some of them had double vision, some had slurred speech or could not swallow, and all of them were men. And most of them had testicular cancers.”
In some cases, the germ cell tumors had yet to be diagnosed, but when these patients’ physicians looked for the cancer, it was found in nearly every case, says Dr. Dubey, adding that in most cases the tumor was in the testicular region, but 15% to 20% of patients developed germ cell tumors in their chest or back.
“So even without knowing the protein, the TIFA pattern itself provided meaningful info,” Dr. Dubey says. “It was such a clean story, and the whole point of us trying really hard to find the protein was we wanted to offer it as a test, even before (KLHL11) was discovered.”
In fact, because the “sparkles” tissue staining pattern had such high clinical specificity when detected and was uniformly found in young adult or middle-aged men who presented with hearing loss, difficulty speaking, and imbalance, the neuroimmunology team flirted briefly with the idea of offering “sparkles” pattern testing through TIFA.
“We’d already put in a lot of resources in antigen discovery and were optimistic we would be able to find the protein, and that way be able to offer a more robust test,” Dr. Dubey says.
When a collaborative effort between scientists from Mayo Clinic and the University of California San Francisco, discovered in 2019 that the “sparkles” pattern was cast by antibodies to KLHL11, Mayo Clinic’s neuroimmunology laboratory team hit the gas on their test development.
“We knew this test was going to be extremely important, and it wasn’t a test that we wanted to wait five years for. We wanted to get this to patients as quickly as possible,” Dr. Dubey says. “This is where we stand out in the crowd — we are the only laboratory in the world offering this testing.”
While TIFA had been the testing means through which affected patients had historically been identified, the methodology lacked the sensitivity to catch all positive cases, says John Mills, Ph.D., co-director of the Neuroimmunology Lab.
John Mills, Ph.D.,
“As Dr. Dubey was discovering the antigen and looking at methodologies to confirm presence of these antibodies, we’d encountered an issue that we didn’t have an easy way to detect the antibodies,” Dr. Mills says. “All the previous methods used during the discovery phase were research methods and weren’t really suitable for testing in a clinical laboratory.”
Because the “sparkles” pattern generated by KLHL11 consists of hundreds of small dots in certain specific regions of the slide, which can be difficult to pick up while our technologists are screening thousands of TIFAs every week, cases were still being missed, Dr. Dubey explains.
“Soon after the (biomarker) discovery we started looking into other methodologies, like ELISA and Western blot, to see if those would be transformed into assay. But they had significant issues and none conformed to our expectations,” Dr. Dubey says.
“With the conundrum of Kelch, we were asking ourselves, ‘How can we test a lot of samples quickly?’” Dr. Mills adds.
Recognizing that automation could enable a thorough and efficient specimen analysis prompted the team to consider using a slide scanner, a semi-automated methodology that eliminates the need to manually assess each assay. Because Mayo Clinic’s Neuroimmunology Laboratory tests about 150,000 biospecimens each year, using a high-throughput testing mechanism was also important to simultaneously evaluate samples from many different patients.
“We started to think about using a high content imaging system in the 96-well plate format,” says Dr. Mills. “We had considered this instrument for other purposes previously and realized that it could work for KLHL11 antibodies. We thought it had a high likelihood of succeeding.”
The Confocal High-Content Imaging System had been widely used in the research arena, but never in a clinical lab environment, Dr. Mills says. “In terms of a reference lab using this testing mechanism, we are the first to do that that I’m aware of, at least in the U.S.”
Through the imaging system, up to 92 patient samples and four control samples can be placed onto each plate. Once scanning begins, the instrument takes about four hours to produce multiple images for each sample, which are then interpreted by the computer, says Dr. Mills.
The computer measures the signal intensity of bound antibodies as well as the amount of KLHL protein present in each cell and generates ratio from this information. If the ratio is above a certain cut-off, the patient is considered “reactive” for the antibody.
“I think this is kind of the first step for our laboratory into what is going to be an emerging field of using artificial intelligence and algorithmic-based models to identify antibody patterns,” Dr. Mills says. “I would consider it automated interpretation of microscope-based images — it was key to launching this test, but also enabled the lab to step into the space, and we do anticipate we’re going to build on it.”
To the neuroimmunologists involved in the Kelch-11 odyssey, the rate at which the KLHL11 clinical test was developed and implemented illustrates the importance Mayo Clinic places on providing the right test to the right patient.
“When Mayo Clinic Laboratories really decides something is a priority and everyone works together as a team and puts all of our resources together, it’s remarkable how quickly things can come together,” Dr. Mills says. “The reality is there’s probably 30 people involved. Everyone made it a priority from all avenues and pushed it forward.”
For patients affected by testicular cancer-associated paraneoplastic encephalitis, the test enables hope for a return to normalcy.
“As we say about stroke, ‘time is brain.’ And I think the same applies for these neuroimmunological diseases,” Dr. Dubey says. “The earlier we can catch these patients and treat them appropriately, the better chance we have for reversing or stabilizing the condition.”
In a recent discovery by Mayo Clinic Laboratories, a novel hemoglobinopathy category was identified and termed epsilon gamma thalassemia. The first instance of the disorder was found in 2017 when an obstetric patient underwent a routine screening for blood-related illnesses such as sickle cell disease and thalassemia. Upon completion of additional tests, doctors found an abnormality they had never seen before.
Mayo Clinic’s cardiac (CV) remote monitoring service uses the compact MoMe Kardia cardiac monitoring device that yields a continuous, 24/7 stream of a patient’s ECG and motion data, no matter their location. Any troubling or burgeoning events are observed virtually the moment they occur, allowing one of Mayo Clinic’s certified rhythm analysis technicians to intervene and facilitate care in near real time. And this is only the beginning; remote patient services are the way of the future, and the future is already here.
Tying together the expertise and curiosity of Mayo Clinic autoimmune neurology researchers with eager patients who have rare disease and are looking for answers, the innovative collaboration benefits both patients affected by MOGAD and scientists on the front lines of discovery.