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Hi, I’m Matt Binnicker, the Director of Clinical Virology and Vice Chair of Practice in the Department of Laboratory Medicine and Pathology at Mayo Clinic. In this month’s Hot Topic, Dr. Curt Hanson will discuss the use of laboratory-based prognostic markers in patients with chronic lymphocytic leukemia or CLL. He will highlight the importance of molecular analyses for IGHV and TP53 sequencing in these patients. I hope you enjoy this month’s Hot Topic, and I want to personally thank you for allowing Mayo Clinic the opportunity to be a partner in your patient’s health care.
Thank you for that introduction, Matt. As Dr. Binnicker mentioned, I will be talking today about the role of immunoglobulin heavy-chain variable (IGHV) sequencing and TP53 sequencing in the evaluation of patients with chronic lymphocytic leukemia (CLL).
I have no disclosures.
Our goals today are, first of all, to understand the use of prognostic markers in CLL patients; second, to highlight the importance of molecular analyses for IGHV and TP53 sequencing in CLL patients; third, to understand how IGHV mutation analysis provides prognostic information in CLL and can help inform clinicians about possible treatment decisions; and, finally, recognizing that TP53 mutations identified by sequencing studies are associated with poor outcomes, since those patients are more likely to be resistant to standard therapeutic regimens.
So, let’s start with a brief overview of CLL. CLL is a neoplasm of small mature B-cells and is the most common leukemia diagnosed in adults. The median age at diagnosis is around 70 years. About 10% of cases occur in patients younger than 55 years of age, and the 5-year survival rate is quite good at over 80%. Its clinical course, however, will vary tremendously from being indolent and slowly progressive to a more progressive course that requires aggressive treatment. It is this variation in clinical course that has driven the development of prognostic markers and new treatment protocols in CLL with the ultimate goal being to identify those who need a more aggressive therapeutic approach versus those in whom a hands-off approach is best. This is why various prognostic markers have been developed over time in CLL. These have included clinical staging systems, serum markers, immunophenotypic markers, and genetic anomalies. Just as important, therapies for progressive disease in CLL have rapidly expanded with significant improvements in overall survival.
While the initial prognostic markers were the Rai and Binet stage-based processes, the next markers were all flow-based. Most of us are familiar with CD38 and ZAP70 as they were the additional prognostic markers in CLL. Unfortunately, neither has stood up as an independent marker over time with both having up to 30% discordance as compared to IGHV mutation status. So, in conclusion, CD38 and ZAP70 cannot be used as a replacement for IGHV mutation status in determining a prognosis and, indeed, we have discontinued doing that testing for patients here at Mayo Clinic. I do want to mention one other flow-based marker, CD49d. Of all flow markers, it is the one that may have a prognostic role. However, it is not currently part of the international prognostic index I’ll be talking about later.
Most are aware of the role that fluorescence in situ hybridization (FISH) plays in the prognosis of CLL. It is important to realize that FISH markers are not diagnostic of CLL as those anomalies can be found in other low‑grade B-cell disorders. In CLL, 13q- is certainly associated with a good prognosis, trisomy 12 is typically associated with an intermediate prognosis, 11q- is associated with a poor prognosis and, finally, 17p- or P53 deletion is associated with a very poor prognosis. It has a relatively low incidence at diagnosis, but a much higher incidence following fludarabine treatment.
Next, I want to talk about the immunoglobulin heavy-chain variable region, or IGHV. There are primarily two possible results with this assay. The first is unmutated IGHV, which is defined as the immunoglobulin heavy-chain sequence from the CLL having less than 2% difference in base pair sequences as compared to a reference germline sequence. An unmutated status is associated with a poorer prognosis, and about 40% of all CLLs will be unmutated at diagnosis. This is in contrast to mutated IGHV, which is defined as the CLL sequence having greater than or equal to that 2% difference from the germline heavy-chain sequence. This status is associated with a much better clinical prognosis. Please note the significant difference in overall survival between these two categories. So, IGHV has a clear association with median survival of CLL patients, and as such is now being used to help clinicians with treatment decisions and to identify patients who may benefit from modern therapies such as BTK inhibitors like ibrutinib.
It’s also important to be aware of the role of sequencing TP53 for point mutations in CLL, which is related but separate from FISH for 17p-. The two have a similar incidence at diagnosis and post-fludarabine treatment, but the important thing to know is one can have point mutations while not having a FISH abnormality and vice versa. Therefore, it’s critical that both assays be done. Both of these are associated with poor outcomes, and patients may be relatively resistant to standard chemotherapy and chemoimmunotherapy regimens. They fare much better when treated with small-molecule inhibitors of BTK, such as phosphatidylinositol 3-kinase or BCL2. Venetoclax is an example of a BCL2 inhibitor in use today. Thus, assessment of both 17p- by FISH and TP53 by sequencing has prognostic value and may help guide therapeutic decisions in routine practice.
This next slide goes through the step-wise process of what our clinicians do when applying risk to clinical decisions in CLL patients. First of all, initial risk assessment is based on FISH and IGHV mutation status. The lower risk, as you can see, would be mutated status with low-risk FISH test and the converse being true for identifying those higher-risk patients. For those low-risk patients, traditional treatment would be with chemoimmunotherapy. If there is intolerance of therapy or progression of disease, or for those patients with higher risk, the use of a BTK inhibitor such as ibrutinib would be strongly considered. Then treatment would move on to the other small-molecule inhibitors if a patient has progression of disease with ibrutinib.
One of the best and most comprehensive studies I’ve seen in CLL has led to development of the International Prognostic Index (CLL-IPI). This study looked at over 3,400 treatment naïve patients as part of an international study. There was external validation using over 800 CLL patients from the Mayo Clinic, and what it came down to is that there were only five parameters that were needed to stratify these patients into four risk groups. These groups clearly separated out based on outcomes and time to treatment. Thus, the CLL-IPI has led to improved clinical staging, which in turn has allowed us to better test novel therapeutics in these high- and very high-risk groups.
These guidelines, which were updated in 2018, are summarized in this table. You can see the recommended tests both at diagnosis and pre-treatment. It’s important to highlight the recommendation that TP53 and IGHV mutation assays should be performed before treating patients with CLL.
Here’s a summary of the CLL-IPI risk classification system. It’s a weighted scoring system based on five risk factors with P53 having the highest score of 4. IGHV and serum beta-2-microglobulin have 2 points, and clinical stage and age have 1. So you can see that the prognostic score can run anywhere from 0 to 10. The very high-risk group is identified as having a score from 7 to 10, and if you look closely at the numbers, you can see that the only way to get to that very high-risk group is by having an abnormality of P53 whether by point mutation or by FISH status. The most likely way to get to a high-risk group is having IGHV and/or beta-2-microglobulin abnormalities. Thus, the recurring theme of today—the importance of IGHV and TP53 molecular studies in our CLL patients.
This Kaplan-Meier survival curve from the Mayo cohort clearly shows the distinct differences in overall survival between the various CLL-IPI risk categories. The very high-risk group has a 3½ year survival for 50% of the population, while the low-risk group has not even reached that threshold after a 13-year follow-up.
So how is this applied in real life care of the CLL patient? This slide outlines a very simplistic application of the CLL-IPI in clinical practice. First of all, I am certainly not recommending this as the dogma in clinical treatment, but it at least gives you an idea about how these risk groups might be applied clinically. For the low-risk group, the classic treatment of CLL is basically “wait and watch” as these patients have a very low likelihood of progression of disease. The intermediate group typically has to be followed more closely, but these patients usually don’t need treatment unless they become symptomatic. It’s the next two groups, the high and very high risk, which typically require treatment. The very high-risk group, which would have a TP53 abnormality, may be treated as part of an experimental protocol as typical chemotherapy or chemoimmunotherapy may not be the best option.
In summary, our knowledge about CLL continues to grow. With the emergence of molecular technologies, it is so critical that the right tests get ordered up front in patients with CLL. In today’s world, it’s more than just doing flow cytometry and making the diagnosis. We need to be sure that the right prognostic tests get ordered in these patients. Hopefully, I introduced you to the CLL-IPI as a relatively simple yet elegant way to stratify patients into appropriate prognostic groups. We know that FISH testing is well established and understood, but we must realize the importance that IGHV mutation analysis has in providing critical prognostic information and how it can help inform clinicians on treatment decisions. And, finally, mutations identified by TP53 sequencing studies are associated with poor outcomes in patients and may be resistant to standard chemotherapy and chemoimmunotherapy regimens.
I thank you for your attention today. Hopefully I’ve been able to provide some new insight in understanding CLL and the critical role laboratorians and pathologists have in the workup and evaluation of these patients.