[Music] what is fish testing how we test is we currently test this routinely in the clinic by the testicle fish which stands for fluorescent inside to hybridization what you see there is a i think it's a beautiful image i've always loved to see fish images what you see in in in the top part the blue that's a myeloma cell it's been uh painted with a dye that makes it look blue so we can only score the myeloma cells and then you see those little dots that you know the the red and the green and we kind of asked the questions okay how many dots do we have there are the dots separate are there dots together and with these questions we can make some determinations you know is there a chromosome that is lost is there an extra copy of chromosomes or two chromosomes together when they should be apart and that's how we we read actually the fish fish results and the bottom part you see a normal neutrophil so if you have had a transplant you know we're waiting for those to come back that's a neutrophil in person so you know you know how they look like and as you know they all have the same genes and chromosomes and that's why the probes go there as well too what are the limitations of fish testing fish testing eye fish interphase fluorescence in situ hybridization is a test with very limited scope you can only look for a variety of markers the good thing about it is that you can get that information on all patients most people do a panel where they look for the translocations to the immunoglobulin region so that test will pick up the 4 14 11 14 14 16 anything to that amino globulin region it normally uses a split apart probe where normally you would have one signal but if there's a break you have three signals one from the normal copy on these two others so that picks up one set of the lesions and then there are copy number abnormalities like 1q gain loss of 17p so if you do the specific translocations plus gain 1q loss of 17p you get most of the lesions that are important for making prognosis the problem with 17p which is where the gene p53 sits is that you can have a deletion of 17p but it not important prognostically at all and it's really the mutations in p53 that count so there's an important message for patients which is if you have 15 20 seventeen b minus which is how it's described don't necessarily think that you have high-risk disease so fish testing is probably the most common genetic test on in multiple myeloma and fish stands for fluorescent in situ hybridization and really what that means is we're able to look at chromosomes that aren't in cells that are dividing and we use a probe that fluoresces and so we can see it with a color and we can stain a cell and we can look to see if that probe is where it's supposed to be or if it's somewhere else and to do that we use two probes of two different colors and we can look to see if they're together or if they're separated and that's how we look for some of the common genetic events in multiple myeloma like the 414 chromosome translocations or the 1114 chromosome translocation who reads the results of fish testing so generally the fish is done by a cytogeneticist although there are some machines that are capable of doing that um it's not that it's not widely done by a machine it's usually done by a person what are the main probes used in fish um the main probes are one to look for chromosome translocations and typically people would look for the 11 14 the 4 14 and the 14 16. and then other probes look for gains or losses of chromosomes and the common one would be loss of chromosome 13. then we look for loss of chromosome 17p and then gains of odd-numbered chromosomes which would be 3 5 7 9 11 15 17 and 19 and gain of chromosome 1q do all labs perform the same fish testing panel with fish testing although there are lots of probes that can be used not every lab uses every probe often there isn't enough material to do that and so there has to be some selection as to what you look for and it may differ from in a newly diagnosed patient and then in a relapse patient and so often you might look for five different things when you do a fish test and then try and draw a conclusion about everything that's happening what does a fish testing report look like the reports are really confusing even for people who do this for a living and it's because there's different ways of reporting it um so for instance they they should tell you the probes that were looked that were examined and they should tell you if it was normal or abnormal but sometimes an abnormal signal doesn't necessarily mean what you think it means and so to give an example the 1114 chromosome translocation is identified when a probe from chromosome 11 goes next to a probe from chromosome 14. and if you see that you call it abnormal but if by chance you had three copies of chromosome 11 which is also quite common that might be reported as abnormal it is because there's three copies but it doesn't mean that there's an 11 14. so the the interpretation of the fish results can be very very tricky for someone who's not trained in it although they there should there should be a summary by the cytogeneticists describing what they think is present when should fish testing be done they should have it done as part of their initial bone marrow biopsy to evaluate multiple myeloma it's part of the revised international staging system for multiple myeloma which is applied to all newly diagnosed patients and in order to stage a patient you need to know whether they have a 4 14 a 14 16 or a deletion of 17p and so it should be done at that point and then when a patient has relapse and it seems as though their disease may have changed character then that would be another important time to reassess because there are some genetic events which are present at the very beginning and stay with the patient throughout the course of their disease such as the chromosome translocations but then there are other events that seem to come on with tumor progression that are associated with perhaps more aggressive disease like a gain of 1q or deletion of 17p and so it's important periodically during the course of a patient's disease to reassess the myeloma to see if it's acquired genetic events associated with disease progression what does it mean to enrich the sample used during fish testing so an important aspect about doing fission multiple myeloma is to be sure that you're actually looking at myeloma cells and not normal cells and so many places will purify out the myeloma cells before doing the fish or other places will look for the presence of light chain genes in the cell that they're looking at kappa or lambda and if you do that then you can have a reliable indication of you know what genetic events are in the myeloma but many places don't do that they just take the whole bone marrow which often by the time it gets to the the fish laboratory is dilute and might have only you know five percent myeloma cells in it and then it can become very hard to know um how many uh of the myeloma cells have any given genetic abnormality now when it comes to chromosome 17p um it's important uh to think that when you're looking at only the myeloma cells so you have to make sure that the lab has done either some sort of selection for the myeloma cells are done cytoplasmic immunoglobulin fish then in that case it may be that only a fraction of the myeloma cells have 17p deletion and there are reports that if the fraction is lower than 30 percent that it is not associated with a poor prognosis but that has has been somewhat controversial and i think in part it's influenced by the by other people who haven't purified out the myeloma cells i think more important is sort of more recent genomic data which is said or shown that if you've got the lesion 17p if you've got a mutation of the gene that we think is critical on 17p and that gene is called p53 and if you've got both the deletion and the mutation then that is really poor prognosis but if you just have the mutation with no deletion or just the deletion with no mutation then that is not a poor prognostic feature and what what that implies is that we really need to start doing sequencing of our newly diagnosed patients so that we can tell if they have both those events can fish testing determine your p53 status the fish testing can really just tell you if that whole big piece of dna is present or it's not present but it can't tell you if there's a point mutation in the gene so to get the point mutation so that's a mutation of a single nucleic acid one base really small the only way to see something like that is to do sequencing of that gene in order to see it and so you can you can never get that detail from fish