Scientists map genetic course from chronic lymphocytic leukemia to Richter syndrome

Each year, up to 1% of patients with chronic lymphocytic leukemia (CLL), a slowly growing blood cancer, see their disease develop into a much more aggressive cancer, a form of lymphoma known as Richter. For the most part, the genomic changes that underlie this metamorphosis and drive it forward have remained obscure, hampering therapeutic progress.

In a new study, scientists from Dana-Farber and the Broad Institute of MIT and Harvard trace these changes in unprecedented detail, revealing for the first time the genomic differences between CLL and Richter disease, molecular pathways by which Richter’s disease emerges and the existence of multiple subtypes of the disease.

The findings, presented Dec. 8 at the American Society of Hematology (ASH) annual meeting and published online in the journal natural medicinenot only opening up what was once a “black box” of molecular change, but paving the way for earlier diagnosis of disease, when treatments may be more effective.

“The treatments for CLL and Richter’s syndrome are very different, so it’s critical that doctors are able to determine, as early as possible, when CLL has ‘crossover’ to become Richter’s disease,” says the Study co-lead author Catherine Wu, MD, of Dana-Farber, the Broad Institute and Brigham and Women’s Hospital. “The traditional method of diagnosing Richter’s disease has a number of shortcomings, which can lead to delays in patients receiving appropriate treatment. Our results in this study promise earlier and more definitive diagnosis based on the molecular composition of tumor cells.

One of the biggest hurdles in diagnosing Richter syndrome is that patients have neither CLL nor Richter cells, but a mixture of both. And unlike CLL, which is diagnosed from a blood samplea formal diagnosis of Richter’s disease requires a biopsy, in which a small piece of tissue is removed and examined under a microscope to detect telltale changes in cell structure and markings.

But because a biopsy collects tissue from a single area, it may find LLC cells but miss the Richter cells lurking right next to it. As a result, a patient may have classic symptoms of Richter’s disease such as swollen lymph nodes, fever, night sweats, but the biopsy – which ultimately determines the diagnosis – points to CLL.

To understand Richter at molecular level and follow its evolution from CLL, the researchers started by fabric samples collected from 52 patients over a period of several years. Samples taken when the patients had CLL were matched with samples taken when they were diagnosed with Richter’s disease. The researchers then performed whole exome sequencingby reading sections of protein-coding DNA in samples.

Since the samples likely contained a mixture of cells, they used computational methods on this sequencing data to estimate the proportion of CLL and Richter cells in each. Knowing the relative levels of different cell types in the samples, they were able to identify the genetic changes that lead to the progression of CLL to Richter disease.

The researchers found a motley assortment of these changes, including mutations in multiple genes, missing or added copies of other genes, duplication of cellular genomes, and “chromothripsis,” a fragmented and random reassembly of entire chromosomes.

“We see myriad differences between CLL and Richter’s disease at the molecular level, with a much more complex genome in Richter’s disease, as well as additional motor events,” notes the study’s co-lead author, Dr. Gad Getz of the Broad Institute and Massachusetts General Hospital. “Additionally, we found that Richter exists in a number of different subtypes.”

The subtypes are distinguished by their molecular signatures, the specific pattern of genomic abnormalities within their cells. These differences at the DNA level suggest that the subtypes arrived by taking different routes to evolve from CLL. “The formation of multiple subtypes can give us insight into the ‘archaeology’ of the disease: what was the molecular composition of CLL before it transformed into one subtype or another? Wu explains.

Being able to identify different Richter subtypes can be clinically useful: patients with some subtypes generally fare better than others, although the current outlook is poor for all patients with Richter. Scientists hope that progress can improve these prospects.

Plasma diagnostics

Once the researchers knew the genomic characteristics of Richter’s disease, they investigated whether the disease could be detected by analyzing DNA in patients’ plasma, the liquid part of blood. They sequenced DNA from 46 plasma samples from 24 patients with Richter’s disease. The samples had been collected over a period of several years, beginning within three years of diagnosis of Richter’s disease and extending through treatment and disease relapse. The researchers then sequenced the free-floating DNA in the samples. “We found that the genomic characteristics of Richter were indeed detectable in plasma,” says Wu.

“We then asked if such changes could be detected before patients were diagnosed with Richter’s disease based on biopsy,” she continues. “For some patients, we clearly detected Richter-related DNA alterations in plasma that had been taken one to ten months before their Richter diagnosis – a time when they had been undergoing treatment for what was presumed to be a aggressive LLC.” The result is that it may become possible to diagnose Richter’s disease with a simple blood test, potentially earlier than it would show on a biopsy and at a stage when it may be easier to treat.

“Current therapies for Richter’s disease are of very limited efficacy, but there is hope that patients may benefit from newer, more effective agents. Clinical trials of these agents and stem cell transplantation may explore that promise,” Wu notes. “By the time Richter’s disease is diagnosed, however, patients may be very ill, in which case transplant or other new therapies may not be an option. can make a crucial difference.”

Learning the molecular characteristics of the Richter researchers led to another discovery. In a significant portion of patients, their Richter cells did not share a genetic background with their CLL cellsmeaning that Richter’s disease arose independently, unrelated to the prior disease.

“In view of future work, we would like to analyze even larger cohorts of patients with SR to obtain a comprehensive characterization of the genomic and microenvironmental landscape of SR; from this, we can discover new robust therapeutic targets as well as refined molecular subtypes brings us closer to applying precision medicine to this disease,” adds Dr. Getz

“Our results suggest that in many patients, the genomic changes in CLL that lead to Richter disease occur before patients develop symptoms of lymphoma,” Wu said. “Being able to trace the transition from CLL in Richter’s disease at the molecular level impacts not only our understanding of the disease, but also, potentially, our ability to treat it and improve patient outcomes.”