A Stanford Medicine-led, international study of hundreds of samples from patients with classic Hodgkin lymphoma (cHL) has shown that levels of tumor DNA circulating in the blood can identify those individuals who are responding well to treatment and those who are likely to experience a disease recurrence. The study results suggest that profiling using circulating tumor DNA (ctDNA) could potentially let some patients who are predicted to have favorable outcomes forgo lengthy treatment.

The study also, surprisingly, revealed that Hodgkin lymphoma—a cancer of the lymph nodes—can be divided into two groups, each with distinct genetic changes and slightly different prognoses. These changes hint at weaknesses in the cancer’s growth mechanisms that could be targeted by new, less toxic therapies.

The international research team, headed by Stanford university professor of medicine Ash Alizadeh, MD, PhD, reported on its findings in Nature, in a paper titled “Distinct Hodgkin lymphoma subtypes defined by noninvasive genomic profiling,” in which the authors concluded, “Collectively, these results support the utility of noninvasive strategies for genotyping and dynamic monitoring of cHL as well as capturing molecularly distinct subtypes with diagnostic, prognostic, and therapeutic potential.”

The idea of establishing molecular profiles of tumors is not new, and the evolution of high-throughput technologies has transformed scientists’ understanding of many cancers the authors stated. And while this is true for non-Hodgkin lymphomas (NHLs), the genomic landscape of classic Hodgkin lymphoma less well understood.

About 8,500 people are diagnosed with Hodgkin lymphoma each year in the U.S. The disease primarily affects people between the ages of 15 and 35 years, and those older than 55 years of age. Hodgkin lymphoma patients are treated using chemotherapy, radiation or a combination of both, and about 89% of patients survive five years or more after their initial diagnosis.

Compared with other cancers, Hodgkin lymphoma has resisted analysis by molecular profiling because Hodgkin lymphoma cells are relatively scarce, even within a large tumor. Unlike many other cancers, Hodgkin lymphoma tumors are made up primarily of immune cells that have infiltrated the cancer, making it difficult to isolate the diseased cells for study. “Published cohorts in cHL are relatively small,  and the profiling methods employed have been limited in both genomic breadth and depth,” the investigators noted. “A major reason for these limitations is the relative paucity of malignant cells within cHL tumors, which typically account for ~1% of the bulk tumor tissue cellularity.”

As Alizadeh further commented, “Compared with other cancers, finding Hodgkin lymphoma cancer cells or cancer DNA to study is like searching for a needle in a haystack. A patient can have a football-sized tumor in their chest, but only about 1% of the cells in the mass are cancer cells, with the remainder representing an inflammatory response to the tumor. This has made it very difficult to find the smoking guns that drive the disease.”

For their reported study Alizadeh and colleagues used an optimized DNA sequencing technique called phased variant enrichment and detection sequencing (PhasED-Seq) to home in on vanishingly rare bits of DNA in a patient’s bloodstream, and identify genetic changes that drive the growth of Hodgkin lymphoma.

Developed at Stanford Medicine in 2021 PhasED-Seq builds on a technique called cancer personalized profiling by deep sequencing, or CAPP-Seq, which was developed in 2014 by Alizadeh and co-senior author Maximilian Diehn, MD, PhD,  professor of radiation oncology and the Jack, Lulu, and Sam Willson Professor, to assess lung cancer levels and response to treatment. In comparison with CAPP-Seq, PhasED-Seq is much more sensitive. “CAPP-Seq could detect as few as one cancer DNA sequence in 10,000 non-cancer DNA sequences,” Diehn explained. “But PhasED-Seq can detect less than one cancer DNA sequence in one million non-cancer DNA sequences.”

The researchers used CAPP-Seq and PhasED-Seq to analyze blood samples from 366 people treated for Hodgkin lymphoma at three medical centers including Stanford Medicine. “This approach offers our first significant look at the genetics of classical Hodgkin lymphoma,” said Alizadeh.

Their goal was to learn more about what drives the cancer and how to make successful treatments even easier for patients. “We typically can cure most patients with one line of therapy,” Alizadeh added. “But we are always trying to figure out less toxic chemotherapeutic agents that are gentler to the bone marrow, lungs and other organs, and ways to more precisely target radiation therapy. And a small minority of patients experience a recurrence that can be challenging to treat successfully.”

Surprisingly, the team was able to detect more cancer DNA in the blood than in the cancer tissue itself, Alizadeh said. “That seemed hard to believe until we had analyzed enough samples to show that it was reproducible.”

The researchers also used machine learning techniques to categorize the different types of genetic changes present in the cancer cells. They found that patients could be separated into two groups: one, termed H1, that predominantly has mutations in several cancer-associated genes implicated in cellular survival, growth and inflammation, and another, termed H2, with a type of genetic change called copy number alterations that affects larger swathes of the genome, subbing in or excising regions of DNA that influence cell growth and cancer.

“We adapted a method from natural language processing to find these two Hodgkin subtypes, and then used a variety of methods to identify key biological and clinical features and to confirm that the subtypes are also seen in other groups of patients,” said co-lead author and instructor of medicine Mohammad Shahrokh Esfahani, PhD.

The first group, which were primarily younger patients, and included about one-half to two-thirds of patients, has a comparatively more favorable outcome. “Cluster H1 tumors comprised ~68% of cases and were dominated by somatic mutations in genes canonically involved in NFκB, JAK/STAT, and PI3K signaling, pathways,” the authors wrote. About 85-90% of individuals in this group survive for three years without disease recurrence, the team found.

The second group, which makes up about one-half to one-third of patients, includes both younger and older patients, and is associated with a less favorable, although still good outcome. . “… cluster H2 tumors, which comprised ~32% of cases, were primarily characterized by a variety of SCNA events as well as mutations in TP53 and KMT2D,” the team continued. About 75% of people in this second group live for at least three years without recurrence the team reported. “Comparing the two clusters, we observed H1 tumors to have a significantly higher somatic SNV mutational burden (P=0.00024), and H2 tumors to have a significantly larger fraction of their genome affected by SCNAs,” the investigators stated in their paper.

Critically, a subset of both groups contained a unique mutation in a gene for the receptor for cellular signaling proteins called interleukin 4 and interleukin 13. “We discovered a new class of mutations in the interleukin 4 receptor gene that enhance a key pathway characteristic to Hodgkin lymphoma,” said co-lead author and postdoctoral scholar Stefan Alig, MD. “These mutations may be indicative of unique vulnerabilities of the tumor that can be exploited therapeutically.”

As the authors reported, “Importantly, unlike transmembrane mutations identified in Non-Hodgkin lymphomas, this novel class of mutations appears uniquely susceptible to drugs targeting IL4R.  Antibodies targeting IL4R or related proteins, which are currently used to treat atopic disease,  could therefore have a future role in the treatment of cHL, perhaps as part of abbreviated chemotherapeutic strategies relying on immunological blockade.”

And of particular interest, the team noted, the truncating IL4R mutations are the only genetic alterations described to date that seem to exclusively occur in cHL, and therefore discriminate between Hodgkin and non-Hodgkin lymphomas.

The researchers also showed that patients who had no detectable circulating tumor DNA in their blood shortly after starting treatment were much less likely to have disease recurrence than those who had even small amounts of residual circulating cancer DNA at the same time point—a distinction researchers had hoped to see, but were unsure about being able to detect even with PhasED-Seq. “Patients with durable remissions experienced more precipitous and sustained ctDNA decreases compared with patients ultimately experiencing disease progression,” the scientists stated. “Specifically, patients with durable remissions achieved significantly lower ctDNA levels at various milestones throughout treatment … Accordingly, ctDNA detection was significantly associated with relapse risk, even at very low levels.”

Diehn added, “I was surprised that we could predict which patients would recur. Even with our ultrasensitive assay there was a significant chance that the signal from the cancer DNA could become undetectable after treatment, even in patients who eventually recurred. But this didn’t happen.”

The authors concluded, “… using PhasED Seq we demonstrate the clinical value of pre- and on-treatment ctDNA levels for longitudinally refining cHL risk prediction, and for detection of radiographically occult minimal residual disease … Collectively, this study contributes to the broader understanding of cHL biology, offers opportunities to improve risk stratification and response assessment, and describes novel targets for precision therapy.”

The researchers seeking to understand more about the biology of Hodgkin lymphoma have one key goal: the improvement of care for patients. Just over 60 years ago, Stanford radiologist Henry Kaplan, MD pioneered the use of targeted radiation to treat Hodgkin lymphoma. The new therapy, delivered by a high-energy linear accelerator Kaplan developed in the 1950s for medical use, was the first step in a Stanford-driven effort to turn the once fatal cancer of the lymph nodes into one that is now highly curable. Soon thereafter, Kaplan was joined by medical oncologist Saul Rosenberg, MD, and the two worked out ways to combine radiation therapy with chemotherapy regimens, including one known simply as the Stanford 5 (named because it was the fifth in a series of gradually less toxic treatments).

“The number of people who experience recurrence is small, but, like Henry Kaplan and Saul Rosenberg, we want to save every one of them,” Diehn said. “They would have been amazed and gratified by these findings, which build upon their important work from decades ago. We look forward to an era in which we can cure every patient with no toxicity.”

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