Presentation

Presenter

Transcript

Introduction

Hi, my name is Patricia Greipp. I am a member of the Division of Hematopathology at Mayo Clinic. I'm a co-director of the Cytogenetics Laboratory there, and I'm also director of the Core Cytogenetics Laboratory in Mayo Clinic.

Overview

Today I'm going to be talking with you about cytogenetic testing for pediatric hematologic malignancies at Mayo Clinic through Mayo Clinic Laboratories. What I'm going to focus on today is fluorescence in situ hybridization, or FISH testing, particularly related to hematologic malignancies. We'll then focus on our offerings for the Children's Oncology Group (COG) testing and clinical trial enrollments for these children with hematologic malignancies. And we'll finish by comparing our standard and our COG orderable cytogenetic tests through Mayo Clinical Laboratories.

Cytogenomic testing: Risk stratification & therapy targets

So, let's start with background. A little background on cytogenomic testing for risk stratification and therapeutic targets, and hematologic malignancies in particular. We'll start out with chromosome analysis, which you may be familiar with. Shown on the right-hand side is a karyotype showing the 22 autosomes and the X and Y chromosomes. This analysis requires dividing cells. It does provide whole genome coverage. However, it's rather low resolution at five megabases. Currently, it is recognized as an integral part of testing for the workup of acute leukemias by the ASH-CAP guidelines and the NCCN guidelines.

We do know that there are limitations with this testing. In particular for B-ALL, this entity tends to have a low mitotic index and a number of the abnormalities are cryptic or not visible by chromosome analysis. For instance, ETV6::RUNX1. So given that this is a rather low-resolution analysis tool, we use it in concert with fluorescence in situ hybridization analysis. This offers higher resolution. Most of our FISH probes are on the order of 100 Kb, some as small as 40 Kb. This does not require dividing cells and is actually performed on interface. This is site-specific testing, which is performed using fluorescence tags hybridized to specific sequences in the genome, for which we can detect monosomies, trisomies, deletions, duplications, and translocations. Shown on the right is a FISH image of two interphase nuclei showing ABL1::BCR fusion. This is representing a 9;22 translocation, common in CML and B-ALL.

When we aren't able to find a primary driver in the acute leukemia, we often turn to microarray analysis. This does not require dividing cells and is performed on extracted DNA. It's quite high resolution on the order of about 10 Kb, and what we can detect are copy number variants and loss of heterozygosity. We cannot detect rearrangements such as translocations. This does provide whole genome coverage, about 2.5 million copy number markers and 750,000 SNPs across the genome. Again, this can't detect balanced rearrangements and independent clones might not be appreciated.

In addition to finding abnormalities that are not visible by conventional chromosome analysis, we can sometimes detect whether a doubled hypodiploid clone, known as a pseudo hyperdiploid clone is present, so microarray analysis is integral for certain entities such as B-ALL. I've also included next-generation sequencing here. This is a robust technology and whole genome sequencing does hold promise for the future. However, currently this is residing currently in the research space primarily. We do have hopes for structural rearrangements and copy number variation detection in the future to enhance our detection of structural rearrangements in hematologic malignancies for pediatric patients.

Mayo Clinic Laboratories (MCL) – A trusted partner

So, moving on, now that we have a feeling for our assays, I'd like to introduce you to the Mayo Clinic's Cytogenetics Laboratory, which has been a forerunner in the field for about 40 years. Each year, thousands of tests are performed in our laboratory from clients around the world. They entrust us with precious blood and bone marrow and tissue specimens sent to us through Mayo Clinic Laboratories in pursuit of diagnostic and prognostic testing in pediatric patients with hematologic malignancies. 

As I mentioned a little bit earlier, we work closely with the Children's Oncology Group (COG), which is the largest clinical trial research organization dedicated exclusively to childhood cancer. They operate clinical trials primarily in North America and New Zealand and Australia, and many pediatric patients are enrolled in clinical trials, and they do require cytogenetic characterization of their acute leukemias as a measure for enrollment in these clinical trials. Mayo Clinic has served for over 25 years as one of a select number of laboratories in the U.S. approved by COG to perform this testing. We perform it for conventional chromosome analysis, FISH analysis, and pediatric bone marrow and peripheral blood specimens. Our dedicated team continually exceeds the COG minimum standard for abnormality detection in chromosome studies, which is currently at 55%, and most recently we achieved 86.4%. And this high detection rate is a credit to our staff, who are skilled in chromosome analysis, which can be quite painstaking. And this allows for more children to receive access to cutting-edge therapies in clinical trials.

Role of cytogenetic characterization

So, what is the role of cytogenetic characterization? Why is it so important? Many hematologic malignancies are driven by structural and numeric rearrangements of chromosomes that drive gene function and cell division. And a great example of this is in B-acute lymphoblastic leukemia/lymphoma. Let's take a closer look. 

WHO Classification: B-lymphoblastic leukemia/lymphoma

The WHO or the World Health Organization classification for B-acute lymphoblastic leukemia/lymphoma is going to be listed here, and what we are going to be talking about are good prognostic outlook, intermediate, and poor prognostic outlook. Prognostic outlook means that these patients are less likely to achieve CR, or complete remission, and are more likely to also relapse down the road. Clinical poor prognostic outlook is rendered for children less than 1 year old and greater than 10 years old, and also those who present with an initial white blood cell count greater than 50,000. 

What I'll present next is good prognostic outlook based on cytogenetic classification, and what you see above is translocation 12;21, which results in ETV6::RUNX1 fusion. This is detectable by FISH and is actually cryptic by chromosome analysis. Hyperdiploidy, with a count of 50 to 65 chromosomes, also renders good prognostic outlook. We can detect this by chromosomes and by FISH.

Intermediate prognostic outlook is rendered by these translocations here listed, translocation 1;19 and 5;14, and additional abnormalities that aren't listed here, not otherwise specified. 

Poor prognostic outlook abnormalities are very important to recognize. For this reason, we focus on KMT2A rearrangements. There are myriad partners for this gene. So, as you see, it's a translocation V or variant with 11q23, which is KMT2A. So, there are over 100 partners of MLL or KMT2A, and we have FISH probes for the most common ones. Also, a poor prognostic outlook is associated with pH-positive or Philadelphia chromosome 9;22, resulting in BCR::ABL1 fusion. Hypodiploidy with a chromosome count of 32 to 43 also is associated with poor prognostic outlook. As I briefly mentioned earlier, this can be masqueraded as a hyperdiploid count when the hypodiploid clone is doubled. So sometimes we utilize a microarray to detect this. Another important entity in this schema is pH-like ALL, and this has a number of drivers which we'll talk about in the next couple of slides. And additionally, iAMP21, or intrachromosomal amplification of chromosome 21.

Diagnostic B-ALL FISH Panel

So, I mentioned that we utilize our chromosome analysis in concert with our FISH panel because many of these abnormalities are cryptic. I briefly mentioned 12;21, which you see listed fourth. It's a 12;21 translocation. And as you see, the KMT2A rearrangement, which I focused on a moment ago, is really important to recognize. This is associated with a very poor prognostic outlook and there are a number of partners associated with KMT2A. It's important to recognize these, and so the most common ones are included as a reflex component of our FISH panel, and they're listed here. As well, IGH rearrangements are associated with an intermediate outlook in B-ALL, but it can also be fused with CRLF2, which renders a poor prognostic outlook. Given that CRLF2 is associated with a poor prognostic outlook, we utilize a break-apart probe upfront in our panel.

So, what you have here is a very comprehensive B-ALL FISH panel. As I mentioned, when we don't find a primary driver, we go on to look for additional abnormalities by array.

pH-like ALL fusion genes

Shown here are the fusion sites for pH-like ALL. The three prime partner genes are listed in bold, and the ones highlighted in gold are included in our pH-like panel. This includes ABL2, PDGFR beta, JAK2, ABL1, CRLF2, and P2RY8. This panel can be ordered separately or as part of our reflex in the comprehensive B-ALL FISH panel. This panel includes an IKZF1 deletion, which often accompanies both pH-positive and pH-negative ALL.

Diagnostic T-ALL FISH Panel

Moving on to T-ALL, we have a number of similar abnormalities that actually are also seen in T-ALL, which includes a 9;22, and KMT2A once again, is included. When we recognize a rearrangement by our break-apart probe we move on to a D-FISH probe or a dual color dual fusion probe, which helps us identify the most common partners. In T-ALL, other drivers include a TRB, which we have reflexed to some common partners, LMO1 and LMO2, and others, as you see listed here, and TRAD. Also, partners listed here. So, this is a very comprehensive panel for our patients with T-ALL.

Diagnostic AML FISH Panel

Moving on to AML, our FISH panel includes common rearrangements of 8;21, which is actually recognizable by chromosomes, and we confirm RUNX1T1::RUNX1 fusion by FISH. Inversion 16 can be cryptic by chromosomes, so we definitely have this as part of our FISH panel. And once again, KMT2A is a player, so we have reflex probes for a D-FISH probe sets listed here. NUP98 is another break-apart probe. This is associated with a poor prognosis. We have PML::RARA associated with a specific type of AML. Patients with acute promyelocytic leukemia have a translocation 15;17 associated with PML:RARA fusion. This is a test that needs to be run in our laboratory rather quickly, and if there is an indication that it needs to be run on a stat order, we are happy to receive telephone calls from our clients and we will report out PML:RARA early to our clients as soon as the testing is completed. We can prioritize this testing to be completed in less than a day, oftentimes.

Moving on in this panel, additional abnormalities that are interrogated by this AML FISH panel include inversion three, which can also present as a balanced translocation 3;3 fusing RPN1 and MECOM or GATA2::MECOM. In KAT6A::CREBBP, associated with 8;16, and the 1;22 translocation, particularly associated with acute megakaryocytic leukemia. You see we additionally have break-apart probe for ETV6, common partner is MNX1. This is associated with a poor prognosis, especially in younger patients with AML.

Pediatric cytogenetic testing orderables at MCL

Our pediatric testing orderables include chromosome testing in blood and bone marrow, listed here as CHRBL and CHRBM. Our FISH testing that we've walked through today is available as AMLPF, BALPF for B-acute lymphoblastic leukemia/lymphoma, and TALPF. This testing is available on the Mayo Clinic Laboratories website. 

Children’s Oncology Group (COG) orderables at MCL

In addition, we've packaged our testing for children who are being enrolled in clinical trials through the COG or the Children's Oncology Group. These specific orderables have different names and include COGBL and COGBM for chromosome testing and FISH testing for acute myeloid leukemia as COGMF, COGBF for B-ALL, and COGTF for T-ALL. This testing is important to send to central review for proper enrollment of patients in these clinical trials, and we'll walk through in the next slide what happens behind the scenes for this testing.

MCL cytogenetic testing for COG trial enrollment

So, Mayo Clinic Laboratories performs cytogenetic testing for this trial enrollment by receiving the specimen from the candidate institution. We perform the testing for chromosome and FISH analysis and array as necessary, and we formulate a report that goes back to the client. In addition, we submit electronically the results to COG for central review of the cytogenetic data. Oftentimes, this may result in a question from COG about particular entities for which we may need to do additional testing to characterize the abnormalities. Once this is achieved in a timely manner, it's reviewed again by central review and moved on to COG trial enrollment. This happens at the client institution, but really important work goes on in the background and this is something that you can be assured will be happening at Mayo Clinic for your patients.

MCL – COG orderables

Here's a listing of the Mayo Clinic Laboratories COG orderables in comparison to the pediatric orderables we talked about a bit earlier, and essentially, they're the same. COGMF for AML is similar to AMLPF, this is for FISH testing. For chromosome testing, COGBM again is for patients being enrolled in COG trials. This is identical to CHRBM for patients who are not being enrolled in COG trials. Additionally, we have chromosomal microarray using different specimens. CMAH is for blood and bone marrow; CMAT for tumor and fresh tissue; and CMAPT is for tissue on paraffin-embedded tissue.

Summary

So, in summary, cytogenetic testing for pediatric hematologic malignancies is critically important for clinical trial enrollment. At Mayo Clinic Laboratories, we are a trusted COG partner for this laboratory testing, and as we've walked through in previous slides, we offer chromosome testing, FISH testing on blood and bone marrow and tissue if necessary, and we also perform chromosomal microarray testing as needed. There's tremendous value at Mayo Clinic Laboratories in terms of what we provide your patients. We provide, as we've walked through, comprehensive cytogenetic testing with a competitive turnaround time listed on the Mayo Clinic Laboratories website. And importantly, we have a very accessible communication team. We talked about the importance of PML::RARA detection by FISH, this can be provided through STAT FISH testing, as communicated to our team, and released by our technologists in an expeditious manner. Again, we offer automatic reflex testing, as demonstrated with the D-FISH probes that we provide for KMT2A, for example, and you can trust that our technologists have a depth of experience. Some have been with us for over 35 years. Pediatric hematology expertise of ABMGG board-certified cytogeneticists and molecular geneticists is available. We're happy to talk with you and we talk with physicians and providers around the country on a daily basis. We look forward to talking with you. Thank you for your time.

Questions?

Contact us: mcleducation@mayo.edu

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