WEBVTT 00:00:08.366 --> 00:00:11.700 Welcome to this video lecture entitled CML, 00:00:12.166 --> 00:00:15.233 FML at day and other Things you should Know. 00:00:15.500 --> 00:00:18.000 Presented by Doctor David E, 00:00:18.000 --> 00:00:20.833 he is the medical director of Connecticut Biology 00:00:20.833 --> 00:00:23.766 AVP laboratories and an assistant 00:00:23.766 --> 00:00:28.266 Clinical professor of pathology at the University of Utah. Doctor. 00:00:28.266 --> 00:00:31.466 He received his medical degree from the University 00:00:31.466 --> 00:00:34.566 of Illinois at Chicago College of Medicine. 00:00:35.100 --> 00:00:38.433 He then completed an anatomic and clinical topology 00:00:38.433 --> 00:00:42.433 residency at Dartmouth-Hitchcock Medical Center, 00:00:42.833 --> 00:00:46.966 and managed the Biology Fellowship at the University of Washington. 00:00:47.566 --> 00:00:52.200 Doctor is board certified in anatomic and clinical pathology and hematology. 00:00:52.500 --> 00:00:56.500 He is the recipient of the Janice Georgi Young Investigator Award 00:00:56.766 --> 00:01:00.533 and the Journey to Repeat Grant for Laboratory Quality Assurance. 00:01:01.066 --> 00:01:05.233 His research interests include minimal residual disease testing, clinical 00:01:05.233 --> 00:01:09.300 flow cytometry and deep learning applications, and flow cytometry. 00:01:09.800 --> 00:01:12.800 I'll now turn the team over to doctor in. 00:01:13.133 --> 00:01:17.233 So, CL slow updates and other things you should know. 00:01:17.933 --> 00:01:21.233 So briefly, we're going to go over the background of CL, the, 00:01:21.500 --> 00:01:25.733 diagnostic criteria for CL also prognostic testing. 00:01:25.733 --> 00:01:27.066 And then there Gnostic testing. 00:01:28.800 --> 00:01:31.600 The learning objectives are before you here 00:01:31.600 --> 00:01:35.533 understanding how to make the diagnosis of CL, differentiating 00:01:35.533 --> 00:01:39.400 between the various prognostic and diagnostic testing out there. 00:01:40.033 --> 00:01:42.433 And then recognizing interference 00:01:42.433 --> 00:01:45.433 of these assays. 00:01:45.933 --> 00:01:47.633 So CL versus 00:01:47.633 --> 00:01:51.366 SL we see these terms used almost interchangeably. 00:01:52.200 --> 00:01:55.200 Clal stands for chronic lymphocytic leukemia. 00:01:55.366 --> 00:01:58.366 And SL stands for small lymphocytic lymphoma. 00:01:59.366 --> 00:02:02.900 Really they're the same disease but they have different manifestations. 00:02:03.333 --> 00:02:06.733 In this Venn diagram, we can see that the vast majority of cases 00:02:06.733 --> 00:02:09.833 are actually c a are actually leukemia cases. 00:02:11.700 --> 00:02:15.300 There's a large subset that's both leukemia and lymphoma. 00:02:15.366 --> 00:02:18.366 S and then a very, very small subset 00:02:18.366 --> 00:02:21.366 that is just SL, just lymphoma. 00:02:22.266 --> 00:02:24.100 This is a very common disease there, 00:02:24.100 --> 00:02:27.100 over 21,000 new cases every year in the US. 00:02:27.333 --> 00:02:30.500 And this doesn't even include the monoclonal B-cell lymphocytes 00:02:30.500 --> 00:02:33.966 versus these are true cases of CL cell. 00:02:34.500 --> 00:02:36.766 This is a very indolent disease. 00:02:36.766 --> 00:02:38.600 85% five year survival. 00:02:38.600 --> 00:02:41.200 And most people who live with this disease, 00:02:41.200 --> 00:02:44.166 there are a lot of people who will live with this disease, either 00:02:44.166 --> 00:02:47.400 with clinical manifestation or subclinical manifestation. 00:02:49.266 --> 00:02:51.066 So the diagnostic criteria 00:02:51.066 --> 00:02:54.233 are for SL nodal involvement. 00:02:54.933 --> 00:02:58.933 And for CLL it's bone marrow and peripheral blood involvement. 00:02:59.266 --> 00:03:02.266 So we use these terms interchangeably often, 00:03:02.933 --> 00:03:07.066 because you will get both nodal involvement and bone marrow involvement. 00:03:07.866 --> 00:03:10.733 And really doesn't change that much in terms of, 00:03:10.733 --> 00:03:13.733 diagnosis and prognosis. 00:03:14.533 --> 00:03:17.266 So let's talk about the morphology, 00:03:17.266 --> 00:03:21.100 the the principal diagnostic criteria for cluster. 00:03:22.333 --> 00:03:27.266 When you get a patient who has, suspected diagnosis of lymphoma, 00:03:27.533 --> 00:03:30.700 you typically do what's called a excision of lymph node biopsy. 00:03:30.833 --> 00:03:33.833 That's the gold standard way of making a diagnosis. 00:03:34.100 --> 00:03:37.466 And on the lymph node, you'll see an a faced architecture with, 00:03:37.700 --> 00:03:41.833 lighter zones, also called proliferation or pseudo proliferation centers 00:03:42.266 --> 00:03:45.266 and darker areas surrounding them. 00:03:45.633 --> 00:03:48.633 The typical morphology is a small cell infiltrate. 00:03:49.000 --> 00:03:53.533 And when we define small in morphology, we're really talking 00:03:53.533 --> 00:03:56.700 about small with respect to a normal resting lymphocyte. 00:03:57.733 --> 00:04:00.966 The, nuclei have a characteristic soccer ball 00:04:00.966 --> 00:04:04.833 like nuclear chromatin with multiple small chromosome centers. 00:04:05.700 --> 00:04:10.733 There's scant cytoplasm and occasional scattered larger forms. 00:04:12.100 --> 00:04:15.600 So here's an image of, of an occasional lymph node biopsy. 00:04:15.600 --> 00:04:21.000 And we can see here that the the node is completely effaced by this small, 00:04:21.566 --> 00:04:24.566 relatively monotonous infiltrate of lymphocytes. 00:04:24.766 --> 00:04:28.933 And at lower power, probably 2 to 4 x, field, 00:04:29.366 --> 00:04:32.166 you can see this, amorphous 00:04:32.166 --> 00:04:35.166 lighter area showing up, 00:04:35.800 --> 00:04:38.800 called the pseudo proliferation center. 00:04:39.066 --> 00:04:41.366 Here's another image from, 00:04:41.366 --> 00:04:42.300 a journal article. 00:04:42.300 --> 00:04:47.400 And we can see at very low power these large kind of, light zones. 00:04:47.700 --> 00:04:49.266 And this is zoomed up on it. 00:04:49.266 --> 00:04:51.466 So these light zone lymphocytes are really 00:04:51.466 --> 00:04:54.433 about the same size, maybe slightly increased. 00:04:54.433 --> 00:04:57.533 And what's causing the, lightness is an increase, 00:04:58.533 --> 00:05:01.533 in spacing between the lymphocytes. 00:05:01.900 --> 00:05:04.500 So occasionally you can see these larger forms 00:05:04.500 --> 00:05:07.500 intermixed with your small mature lymphocytes. 00:05:09.100 --> 00:05:12.100 So these are characteristic findings of, 00:05:12.466 --> 00:05:15.466 a cell in a lymph node. 00:05:15.466 --> 00:05:17.500 Now, in a bone marrow 00:05:17.500 --> 00:05:20.500 or in, smear, you can see, 00:05:21.500 --> 00:05:22.533 smudge cells. 00:05:22.533 --> 00:05:25.600 And, oftentimes you'll have to I'll be minute 00:05:25.600 --> 00:05:29.600 these slides in order to get nice nuclear morphology again. 00:05:30.000 --> 00:05:30.400 Smear. 00:05:30.400 --> 00:05:33.400 You can see soccer ball like nuclear chromatin. 00:05:33.800 --> 00:05:35.400 So this is from the ash image Bank. 00:05:35.400 --> 00:05:37.900 And you can see this characteristic, 00:05:37.900 --> 00:05:39.933 intermediate, small to intermediate sized 00:05:39.933 --> 00:05:42.933 lymphocytes with chroma centers. 00:05:43.266 --> 00:05:46.900 Perhaps nuclei like scant pale blue cytoplasm 00:05:46.900 --> 00:05:51.433 or a scant blue cytoplasm at mixed with 00:05:52.600 --> 00:05:55.600 perhaps more normal appearing lymphocytes. 00:05:55.766 --> 00:05:59.400 In this image you'll see these very high NC ratio cells. 00:06:00.333 --> 00:06:02.533 A couple of basket cells or smudge cells. 00:06:02.533 --> 00:06:05.633 And in this example you definitely see numerous smudge cells. 00:06:06.000 --> 00:06:10.166 So these are characteristic findings of CL in the peripheral blood. 00:06:11.633 --> 00:06:12.533 So this is from an 00:06:12.533 --> 00:06:15.700 interesting paper that did morphologic, analysis on, 00:06:16.733 --> 00:06:19.766 thousands of closely all cells in the peripheral blood. 00:06:20.333 --> 00:06:23.333 And what it's trying to show you is, 00:06:24.933 --> 00:06:28.033 IBM and really makes a difference in terms of the morphology, 00:06:28.033 --> 00:06:30.466 the characteristic morphology of these cells. 00:06:30.466 --> 00:06:34.066 So I included this slide just because it gives you numerous examples. 00:06:34.066 --> 00:06:38.400 If you're trying to, get into your mind, what does a class cell look like. 00:06:39.366 --> 00:06:43.200 And in this case, when we put albumin 00:06:43.200 --> 00:06:46.200 into the, 00:06:46.800 --> 00:06:47.566 fluid 00:06:47.566 --> 00:06:50.166 into the peripheral blood and then smear it out, 00:06:50.166 --> 00:06:53.533 you get a definite change in the cell morphology. 00:06:54.266 --> 00:06:56.533 These cells become much more, small, 00:06:56.533 --> 00:06:59.533 much more characteristic soccer ball nuclei. 00:06:59.800 --> 00:07:02.800 You have the larger proliferating forms that mixed. 00:07:02.800 --> 00:07:05.733 But for the most part, this is the morphology that 00:07:05.733 --> 00:07:09.200 when people describe soccer ball like nuclear chromatin and cloth, 00:07:09.233 --> 00:07:10.433 this is what they're talking about. 00:07:12.466 --> 00:07:14.066 So we can see that, 00:07:14.066 --> 00:07:17.300 without the albumin, all these cells look much larger. 00:07:17.566 --> 00:07:20.100 You may even be tempted to consider them. 00:07:20.100 --> 00:07:21.833 Monocytes. 00:07:21.833 --> 00:07:26.400 But really, you want to be looking for this type of nuclear morphology, 00:07:26.400 --> 00:07:29.633 and that's really associated with LV mediated slides. 00:07:31.466 --> 00:07:34.200 Now, some of you may be familiar with cell division. 00:07:34.200 --> 00:07:37.033 This is an automated, smear technique. 00:07:37.033 --> 00:07:39.800 Uses a different staining. Protocol. 00:07:39.800 --> 00:07:43.166 And so the morphology is oftentimes very different compared 00:07:43.166 --> 00:07:46.500 to what we're used to with the ordinary right game sustain. 00:07:47.500 --> 00:07:50.500 These cells are much more, 00:07:50.633 --> 00:07:54.700 they're hyper chromatic, almost, at high power. 00:07:54.700 --> 00:07:58.033 And maybe if you're looking at it, with your own microscope, 00:07:58.233 --> 00:08:01.300 you can actually see, condensed chromatin. 00:08:01.300 --> 00:08:06.366 But oftentimes these just look like a sheet of darkness with scant cytoplasm 00:08:06.733 --> 00:08:12.166 and really, the clue would be seeing, these, smudge cells scattered about. 00:08:14.466 --> 00:08:15.300 So the immuno 00:08:15.300 --> 00:08:18.300 phenotype of CL cell is very characteristic. 00:08:18.300 --> 00:08:21.300 It typically has retained Cd19, 00:08:21.766 --> 00:08:24.466 and then a decreased expression of CD 00:08:24.466 --> 00:08:27.466 20 low to intermediate CD five, 00:08:27.466 --> 00:08:30.900 usually less than the level of background T cells. 00:08:30.900 --> 00:08:34.100 But sometimes it could actually raise to the level of background T cells 00:08:34.633 --> 00:08:38.700 will have decreased CD 22 decreased to absent CD 79 B 00:08:39.000 --> 00:08:43.566 and apparent expression of CD 200, CD 23, CD 43. 00:08:43.800 --> 00:08:46.800 Loss of FMC seven and decreased CD 81. 00:08:47.433 --> 00:08:50.700 These are flow markers and you can actually do them by image. 00:08:50.700 --> 00:08:51.966 Has chemistry. 00:08:51.966 --> 00:08:57.333 Of note the immunohistochemical markers left one is typically positive. 00:08:57.333 --> 00:09:00.566 And then cyclin D1 is negative. 00:09:00.866 --> 00:09:04.533 Although it can be weakly positive in the proliferation centers. 00:09:06.600 --> 00:09:09.600 So let's talk about flow cytometry. 00:09:09.666 --> 00:09:12.000 This is considered the fastest and cheapest way 00:09:12.000 --> 00:09:15.800 to make the diagnosis of cloud and improves flow cytometry. 00:09:15.800 --> 00:09:18.100 Is using fluorescent. 00:09:18.100 --> 00:09:21.266 Fluorescently labeled antibodies bound to a single cell. 00:09:22.000 --> 00:09:25.833 So what we have here is, laser that excites a fluorophore attached 00:09:25.833 --> 00:09:30.900 to an antibody that binds to a surface marker that defines a cell population. 00:09:31.433 --> 00:09:35.533 And when this laser hits this fluorophore at the four for remits, 00:09:36.200 --> 00:09:39.766 signal at a longer wavelength, and this fluorescence signal 00:09:39.766 --> 00:09:43.400 is considered proportional to the quantity of this target molecule 00:09:43.400 --> 00:09:45.833 that we're looking for. In this case, CD 20. 00:09:47.533 --> 00:09:50.733 So these are dot plots that we can oftentimes 00:09:50.733 --> 00:09:53.733 see in a diagnostic case of Costello. 00:09:53.966 --> 00:09:56.633 Again CD 19 is retained. 00:09:56.633 --> 00:10:00.500 The CD 20 is low or decreased compared to a 00:10:01.700 --> 00:10:04.700 background population of of B cells. 00:10:05.400 --> 00:10:10.500 There's this variable expression of CD 20 and kind of low. 00:10:10.500 --> 00:10:11.133 In this case, 00:10:11.133 --> 00:10:15.433 the CD five is almost the same intensity as the background T cells colored red. 00:10:15.433 --> 00:10:17.033 Here. 00:10:17.033 --> 00:10:20.366 The light chain expression is dimmer to absent 00:10:20.366 --> 00:10:24.366 compared to normal B cells, which would sit up here and up here. 00:10:25.133 --> 00:10:27.800 And then we can see in this population aberrant. 00:10:27.800 --> 00:10:30.933 Co-expression of CD 23 and CD 00:10:31.033 --> 00:10:34.000 200 and low level CD 38, 00:10:34.000 --> 00:10:37.000 which we'll talk about later. 00:10:37.000 --> 00:10:39.033 Now, there's been questions about whether light 00:10:39.033 --> 00:10:42.033 chains are useful in the diagnosis of this disease. 00:10:42.200 --> 00:10:45.033 If the answer is yes or no, 00:10:45.033 --> 00:10:49.400 light chains can be low to negative and in many cases of class. 00:10:49.800 --> 00:10:52.800 And so what you'll see in this particular case 00:10:53.066 --> 00:10:56.033 is a light chain negative classical. 00:10:57.033 --> 00:10:59.500 And it can actually look like it's, it's, 00:10:59.500 --> 00:11:03.166 following the diagonal, pattern that you consider, 00:11:04.200 --> 00:11:06.066 nonspecific binding. 00:11:06.066 --> 00:11:09.166 But again, this population, when you look at it's such subset 00:11:09.166 --> 00:11:12.600 marker expression is 200 positive and 23 positive 00:11:12.933 --> 00:11:17.633 has decreased to CD 20 expression compared to your normal B cells 00:11:18.133 --> 00:11:21.300 and lower CD five than your normal T cells. 00:11:22.300 --> 00:11:24.433 So in this case it's still diagnostic. 00:11:24.433 --> 00:11:27.433 But it's light chain negative. 00:11:27.900 --> 00:11:29.966 Now rarely but 5 to 10% 00:11:29.966 --> 00:11:32.966 of cases will actually have by tonality. 00:11:33.133 --> 00:11:35.100 That is to say your class. 00:11:35.100 --> 00:11:38.766 Our population will have both kappa and lambda light chains. 00:11:39.000 --> 00:11:43.466 And oftentimes these subsets will have varying expression of different markers. 00:11:43.900 --> 00:11:47.300 So in this particular case we see both kappa and lambda light 00:11:47.300 --> 00:11:52.000 chain expression among your five positive 20 dim CL cells. 00:11:52.933 --> 00:11:55.466 And so these the Kappas are colored 00:11:55.466 --> 00:11:58.466 blue, the lambdas are colored red. 00:11:58.500 --> 00:12:01.900 So you can see here your normal B cells are colored here. 00:12:01.900 --> 00:12:04.900 And then your abnormal CL cells are colored here. 00:12:05.100 --> 00:12:06.466 They look like they're poly typically. 00:12:06.466 --> 00:12:09.900 But when you look at their subset expressions you can see, 00:12:10.400 --> 00:12:14.100 oh you're 223 positive subsets 00:12:14.100 --> 00:12:17.100 have kappa and lambda light gene expression. 00:12:17.333 --> 00:12:20.133 Your 200, positive 23 negative 00:12:20.133 --> 00:12:23.133 subset has only lambda light chain expression. 00:12:23.333 --> 00:12:26.500 And vice versa. Your 23 positive 00:12:26.500 --> 00:12:30.466 only subset has, predominantly lambda light chain expression. 00:12:33.933 --> 00:12:35.500 So moving on, 00:12:35.500 --> 00:12:39.600 the diagnostic criteria for class L, defined by the W.H.O. 00:12:39.600 --> 00:12:43.200 these days is, greater than 5000 00:12:43.200 --> 00:12:46.200 neoplastic cells per microliter. 00:12:47.033 --> 00:12:50.333 Morphologically, this is quite difficult to do because separating out 00:12:50.333 --> 00:12:53.333 normal lymphocytes from neoplastic lymphocytes can be a challenge. 00:12:54.000 --> 00:12:57.066 On the other hand, if you have a smear where you have, 00:12:57.666 --> 00:13:00.666 numerous like 30,000 white blood cells, 00:13:01.200 --> 00:13:05.300 and phenotypic evidence of class yellow, then the diagnosis is trivial. 00:13:05.466 --> 00:13:08.366 You're almost assuredly greater than this, 00:13:08.366 --> 00:13:11.366 five paper microliter, cut off. 00:13:11.666 --> 00:13:15.133 Now, by flow cytometry, we can accurately determine 00:13:15.133 --> 00:13:18.633 the percentage of neoplastic cells out of all white blood cells. 00:13:19.033 --> 00:13:22.666 And so getting a percentage of neoplastic cells is quite easy. 00:13:23.500 --> 00:13:25.833 Getting a cell concentration is trickier. 00:13:25.833 --> 00:13:28.833 And there are different ways to go about this. 00:13:28.866 --> 00:13:31.766 One way is to include a bead standard when you're, 00:13:32.833 --> 00:13:34.000 putting this thing through the flow. 00:13:34.000 --> 00:13:35.266 Cytometer. 00:13:35.266 --> 00:13:38.933 But that requires an extra channel and there's an extra step involved. 00:13:40.133 --> 00:13:41.933 During the testing, 00:13:41.933 --> 00:13:45.766 the other common way to do things is to multiply, 00:13:46.500 --> 00:13:51.700 your white blood count by the percentage of neoplastic cells to get a a 00:13:51.700 --> 00:13:54.700 a absolute neoplastic cell count. 00:13:56.666 --> 00:14:00.133 So what about cases with less than 5000 cells for microliter, 00:14:00.500 --> 00:14:03.566 these are called monoclonal B cell lymphocyte ptosis. 00:14:03.866 --> 00:14:06.866 It's considered a CL precursor. 00:14:07.200 --> 00:14:09.600 And studies have actually indicated 00:14:09.600 --> 00:14:12.600 that the count of these things actually matters. 00:14:12.600 --> 00:14:17.700 So for what's called high count CL or high count ML greater than 2.5 00:14:17.700 --> 00:14:21.600 K per microliter there's about a 2% rate of transformation to. 00:14:21.600 --> 00:14:25.300 Clearly, on the other hand, under two and a half K 00:14:25.566 --> 00:14:29.700 per microliter if there's less than 1% rate of transformation, see how? 00:14:30.466 --> 00:14:34.333 Now, just to let you know, some people use the term monoclonal 00:14:34.366 --> 00:14:38.400 B cell office I ptosis is a generic term for any small monoclonal 00:14:38.400 --> 00:14:42.700 B cell population with any type of phenotypic abnormality. 00:14:43.666 --> 00:14:45.866 But for the purposes of this talk, 00:14:45.866 --> 00:14:48.500 a mouse really mean a class, 00:14:48.500 --> 00:14:51.500 like population. 00:14:51.933 --> 00:14:55.900 So let's talk about this entity monoclonal B-cell for psychosis. 00:14:56.233 --> 00:14:59.333 It's really rather rare in young people. 00:15:00.066 --> 00:15:03.433 One study has shown that under the age of 40, you only see it 00:15:03.766 --> 00:15:06.766 in one out of 365 individuals. 00:15:07.333 --> 00:15:10.066 And this is using a very high sensitivity assay. 00:15:10.066 --> 00:15:13.900 So 0.001 sensitivity, flow assays, 00:15:14.600 --> 00:15:17.600 as you get older at the incidence shoots up. 00:15:17.700 --> 00:15:21.400 So amongst 80 year olds there's about a 20% incidence. 00:15:22.100 --> 00:15:25.100 And interestingly there's an a genetic linkage. 00:15:25.566 --> 00:15:30.233 So if you have a family member with CLO you can have a 17 time 00:15:30.233 --> 00:15:35.200 a 17 fold increase in the incidence of males, even in young individuals. 00:15:36.000 --> 00:15:39.266 So that suggests that this is a precursor lesion 00:15:39.466 --> 00:15:41.366 and that there is some kind of genetic, 00:15:42.600 --> 00:15:45.466 cause, genetic relationship 00:15:45.466 --> 00:15:48.466 that leads to these kindred class. 00:15:51.000 --> 00:15:52.266 The other issue 00:15:52.266 --> 00:15:56.366 with CL cell is that you have to exclude other entities. 00:15:56.933 --> 00:16:01.933 So amongst the B-cell non-Hodgkin lymphoma with CD five expression 00:16:02.366 --> 00:16:06.233 class, ileal and mantle cell and farmers are the prototypical examples. 00:16:06.933 --> 00:16:11.333 Very rarely you can have marginal zone lymphomas with five expression 00:16:12.000 --> 00:16:14.766 even rarer than that you can have five 00:16:14.766 --> 00:16:17.600 five co-expression in follicular and farmers. 00:16:17.600 --> 00:16:18.700 And here is cell lymphoma. 00:16:18.700 --> 00:16:22.766 These are case reports that are well described at this point. 00:16:23.466 --> 00:16:26.933 So in reality, if you have a five positive B-cell 00:16:26.933 --> 00:16:31.633 non-Hodgkin lymphoma, the principal differential consideration 00:16:31.633 --> 00:16:35.666 apart from SML is to exclude mantle cell lymphoma. 00:16:36.633 --> 00:16:39.966 And even within the Nccn guidelines, they recommend 00:16:40.200 --> 00:16:43.066 using immunohistochemistry against cyclin 00:16:43.066 --> 00:16:46.066 D1 or Sox 11 or, 00:16:46.500 --> 00:16:48.333 translocation 1114 fish 00:16:48.333 --> 00:16:52.400 to exclude the cyclin D1 IgG translocation. 00:16:53.866 --> 00:16:57.200 And this is because Misdiagnosing mantle cell and pharma 00:16:57.200 --> 00:17:01.266 CLL is prognostic and diagnostically a bad thing. 00:17:01.266 --> 00:17:04.266 This is not one of the the, the 00:17:04.300 --> 00:17:07.700 the treatments for these two entities is very different. 00:17:08.466 --> 00:17:11.700 And so we don't want to mix up these entities. 00:17:11.700 --> 00:17:15.966 And doing the confirmatory testing to exclude mantle cell and pharma is 00:17:16.233 --> 00:17:19.233 is almost standard of care. 00:17:20.366 --> 00:17:23.366 So let's talk about the natural history of disease there. 00:17:23.466 --> 00:17:26.766 As we said before, over 21,000 diagnoses a year 00:17:27.033 --> 00:17:30.033 and about 5000 deaths due to clear. 00:17:30.500 --> 00:17:33.233 So what this means is that most people die 00:17:33.233 --> 00:17:36.233 with CLO not of color. 00:17:36.300 --> 00:17:39.000 It's not to say that classical is a nice disease 00:17:39.000 --> 00:17:43.266 that has significant morbidity and more, there's significant more, bility, 00:17:43.666 --> 00:17:46.600 typically fatigue due to side opinions, mass effects 00:17:46.600 --> 00:17:50.766 due to lymphadenopathy, spinal magli sometimes. 00:17:51.166 --> 00:17:55.500 So it's not something that you would want, but, 00:17:56.266 --> 00:17:59.266 as diseases go, it's probably one of the better ones to get. 00:18:00.933 --> 00:18:03.933 So how did things go south in class? 00:18:04.066 --> 00:18:05.700 Well, 00:18:05.700 --> 00:18:07.633 one of the first, things 00:18:07.633 --> 00:18:10.633 that you might see is marked leukocytes. 00:18:10.800 --> 00:18:14.600 So you can see, white counts, neoplastic white counts 00:18:14.933 --> 00:18:17.700 of greater than 100 K per microliter. 00:18:17.700 --> 00:18:22.433 Widespread disseminated lymphadenopathy is also common when things, progress. 00:18:22.966 --> 00:18:26.266 And then finally there's this entity called Richter transformation. 00:18:27.133 --> 00:18:29.433 This is, diffuse large B-cell lymphoma 00:18:29.433 --> 00:18:32.433 that have arisen from a CLL 00:18:33.000 --> 00:18:35.633 and about 2 to 10% of CL cases. 00:18:35.633 --> 00:18:40.366 And up here, the prognosis of Richter transformation is dismal. 00:18:41.466 --> 00:18:44.366 Typically, these patients die within 1 to 1 and, 00:18:44.366 --> 00:18:47.166 1 to 2 years. 00:18:47.166 --> 00:18:48.900 And so 00:18:48.900 --> 00:18:52.966 as clinicians and physicians, we really want to predict 00:18:53.833 --> 00:18:57.833 who of these patients that we see are going to die of this disease. 00:18:57.833 --> 00:18:59.833 And what can we do to change this outcome. 00:19:01.833 --> 00:19:05.700 So prognostic Lee, not all class are created alike. 00:19:05.700 --> 00:19:08.300 We know some classes are going to do very well. 00:19:08.300 --> 00:19:11.133 These patients are going to die with disease. 00:19:11.133 --> 00:19:14.033 And then some classes are going to progress very quickly. 00:19:14.033 --> 00:19:16.633 And patients will die of disease. 00:19:16.633 --> 00:19:19.633 So numerous different prognostic markers 00:19:19.633 --> 00:19:22.633 have been developed over the past 20 plus years. 00:19:23.833 --> 00:19:26.233 Immunoglobulin heavy chain mutation status 00:19:26.233 --> 00:19:29.233 has been one of the strongest prognostic 00:19:30.300 --> 00:19:32.266 markers. 00:19:32.266 --> 00:19:36.633 TP 353 mutations are a common theme among neoplasia. 00:19:36.633 --> 00:19:40.500 Is mutated Tp53 mutation 00:19:40.800 --> 00:19:43.566 or sorry, mutated Tp53 00:19:43.566 --> 00:19:46.300 loci are correlated with bad prognosis 00:19:46.300 --> 00:19:50.333 because we've lost tumor suppressor activity, karyotype. 00:19:50.333 --> 00:19:55.566 So complex types are oftentimes bad and and myriad of diseases. 00:19:55.566 --> 00:19:57.800 And that holds true in CLL. 00:19:57.800 --> 00:19:59.400 And then finally cytogenetics. 00:19:59.400 --> 00:20:02.966 So there have been recurrent set of genetic abnormalities 00:20:03.433 --> 00:20:05.900 that are associated with worse prognosis. 00:20:05.900 --> 00:20:08.633 And this has been known for at least 20 years now. 00:20:10.466 --> 00:20:11.533 So everything I'm talking 00:20:11.533 --> 00:20:14.533 about is straight out of the Nccn guidelines. 00:20:14.766 --> 00:20:19.133 If you're interested in, in, more depth about prognostic 00:20:19.133 --> 00:20:22.133 and thorough Gnostic testing and see how all, 00:20:23.166 --> 00:20:26.700 one of the big determinants is stage versus grade. 00:20:27.133 --> 00:20:31.633 So stage is the degree of anatomic involvement. 00:20:31.633 --> 00:20:34.500 And it's correlated with worse outcomes. 00:20:34.500 --> 00:20:37.533 So more disseminated disease has higher stage. 00:20:37.766 --> 00:20:40.766 And that leads to worse outcomes 00:20:41.233 --> 00:20:43.400 grade as a morphologic finding. 00:20:43.400 --> 00:20:46.566 So stage is done by clinicians on the floor. 00:20:46.866 --> 00:20:50.366 Grade is performed by pathologists under the microscope. 00:20:50.966 --> 00:20:54.666 And these are morphologic findings that are correlated with worse outcomes. 00:20:56.066 --> 00:20:58.800 So for for example, in some diseases 00:20:58.800 --> 00:21:01.900 high mitotic activity is correlated with worse outcome. 00:21:04.466 --> 00:21:06.000 This the Binay 00:21:06.000 --> 00:21:09.300 and Rise systems are the most published 00:21:09.300 --> 00:21:13.333 and most validated staging systems for CLL cell. 00:21:14.700 --> 00:21:16.533 Generally speaking, 00:21:16.533 --> 00:21:18.933 disseminated disease is bad. 00:21:18.933 --> 00:21:21.600 Cytokines are bad. 00:21:21.600 --> 00:21:24.900 Spleen or magli organo magli bad. 00:21:25.600 --> 00:21:28.433 Gnostic significance. 00:21:28.433 --> 00:21:31.433 Whereas if you have, 00:21:32.300 --> 00:21:33.633 if you don't have those things, 00:21:33.633 --> 00:21:37.966 then your risk status is, decidedly lower in the Binay system. 00:21:38.166 --> 00:21:42.933 It's really concentrated on your hemoglobin level and platelet level. 00:21:42.933 --> 00:21:45.966 So, degree of bone marrow infiltration, really. 00:21:46.400 --> 00:21:49.266 And then, number of, enlarged areas. 00:21:49.266 --> 00:21:53.066 So number of, of, sites of ad and apathy. 00:21:55.433 --> 00:21:58.433 And so you can see in Binay class, 00:21:58.833 --> 00:22:03.166 Binay stage three, really your hemoglobin and platelets are under, 00:22:04.433 --> 00:22:07.800 what, ten grams and and 100 K per microliter. 00:22:08.100 --> 00:22:11.100 And you have, enlarged lymph nodes. 00:22:14.100 --> 00:22:15.566 The immunoglobulin heavy 00:22:15.566 --> 00:22:18.566 chain mutation, status, 00:22:18.566 --> 00:22:20.666 has been known for a very long time 00:22:20.666 --> 00:22:23.800 to be a strong prognostic marker for CLL. 00:22:24.633 --> 00:22:28.233 At the UN, mutated, situation at the UN, mutated 00:22:29.200 --> 00:22:31.833 loci are correlated with worse prognosis. 00:22:31.833 --> 00:22:34.833 And this is held up in numerous studies. 00:22:34.900 --> 00:22:38.300 So far, so much so that some people even, propose 00:22:38.300 --> 00:22:41.700 that these are really two biologically different diseases. 00:22:42.766 --> 00:22:47.400 The mutated IgG Hvy, cases have longer prognosis, 00:22:47.633 --> 00:22:51.666 approximately 293 months compared to 117 months. 00:22:53.233 --> 00:22:54.900 So what is the, 00:22:54.900 --> 00:22:57.600 immunoglobulin heavy chain variable region? 00:22:57.600 --> 00:23:00.566 If you recall back to immunology, your immunoglobulins 00:23:00.566 --> 00:23:03.566 have a heavy chain and light chain component. 00:23:04.400 --> 00:23:07.700 These heavy chains have variable regions, 00:23:07.700 --> 00:23:11.466 followed by your, joining regions and then a constant region. 00:23:12.200 --> 00:23:15.200 And when immunoglobulins are created, 00:23:15.300 --> 00:23:19.833 these are randomly selected and recombined to form a new VG. 00:23:20.766 --> 00:23:21.866 Section. 00:23:21.866 --> 00:23:24.866 After this VG recombination, 00:23:24.900 --> 00:23:29.366 there's this additional step called somatic hyper mutation where this vd 00:23:29.500 --> 00:23:32.500 j is randomly mutated 00:23:32.600 --> 00:23:35.866 in order to increase your immunoglobulin diversity. 00:23:37.766 --> 00:23:39.266 And so, 00:23:39.266 --> 00:23:41.966 tumors that have biologically undergone 00:23:41.966 --> 00:23:44.966 this hyper mutation 00:23:45.166 --> 00:23:48.000 are considered more mature, 00:23:48.000 --> 00:23:52.500 so to speak, and, and have better prognosis compared to cases 00:23:52.800 --> 00:23:58.200 where this VD, DG, is virgin, there's no recombination going on. 00:23:58.200 --> 00:24:01.200 And so it suggests that it's a more primitive, 00:24:01.266 --> 00:24:04.266 stem cell origin. 00:24:04.600 --> 00:24:05.700 So how do we do some, 00:24:05.700 --> 00:24:08.700 how do we measure somatic hyper mutation? 00:24:09.400 --> 00:24:10.666 Well, we do sequencing. 00:24:10.666 --> 00:24:13.900 You have to sequence the argv locus 00:24:14.500 --> 00:24:17.500 and compare it to all known, 00:24:17.766 --> 00:24:21.000 argv, loci out there, of which 00:24:21.000 --> 00:24:24.166 there are probably 70 to 80. 00:24:26.100 --> 00:24:29.500 The way we do it here at R is Sanger sequencing, 00:24:29.866 --> 00:24:32.866 where you sequence that entire, 00:24:33.166 --> 00:24:35.566 flanking region, other ways to do it. 00:24:35.566 --> 00:24:38.566 Our next generation sequencing. 00:24:38.600 --> 00:24:41.333 Either way, there's some bioinformatics involved 00:24:41.333 --> 00:24:44.766 where you have to identify what is the closest, 00:24:45.733 --> 00:24:48.600 v, locus 00:24:48.600 --> 00:24:51.600 and mutate it and then measure the difference. 00:24:52.033 --> 00:24:55.666 So in our own laboratory, we've determined that greater 00:24:55.666 --> 00:24:58.666 than 2 to 3% differences in the base pair 00:24:58.666 --> 00:25:03.400 sequence compared to a germline IG five is considered mutated, 00:25:03.566 --> 00:25:07.300 whereas below that one 0 to 2% 00:25:07.533 --> 00:25:10.533 is considered UN mutated. 00:25:12.000 --> 00:25:15.000 In terms of genetic prognostic markers, 00:25:15.666 --> 00:25:18.166 there are 00:25:18.166 --> 00:25:19.900 far there are three 00:25:19.900 --> 00:25:23.600 there for major genetic markers. 00:25:23.600 --> 00:25:25.800 And then the normal karyotype. 00:25:25.800 --> 00:25:29.366 So 13 Q has the best prognosis up here. 00:25:30.200 --> 00:25:33.233 And £0.17 deletions, which by the way 00:25:33.233 --> 00:25:38.166 is where the Tp53 mutation, Tp53 tumor suppressor 00:25:38.166 --> 00:25:43.066 gene lives is down here and everything else is in between. 00:25:43.066 --> 00:25:46.466 So this is 13 Q this is a normal karyotype 00:25:46.700 --> 00:25:49.700 which looks very much like a 12 q. 00:25:50.233 --> 00:25:51.733 Trisomy. 00:25:51.733 --> 00:25:54.366 This is the 11 q or 00:25:55.500 --> 00:25:56.100 it's the 00:25:56.100 --> 00:25:59.866 Italian Tasia mutation site deletion. 00:25:59.866 --> 00:26:02.866 And then this is Tp53 deletion. 00:26:03.666 --> 00:26:07.200 So you can see there's a wide breadth in, prognoses, 00:26:07.833 --> 00:26:10.833 depending on your genetic profile. 00:26:12.333 --> 00:26:15.333 But I suppose cytometry, there are a couple of, 00:26:16.066 --> 00:26:17.700 prognostic markers. 00:26:17.700 --> 00:26:19.766 Zap 70 00:26:19.766 --> 00:26:22.600 has associated with it for mutation status. 00:26:22.600 --> 00:26:24.500 And so, 00:26:24.500 --> 00:26:27.133 if you don't have the ability to do this mutation 00:26:27.133 --> 00:26:30.133 status testing, it could be a surrogate marker. 00:26:30.400 --> 00:26:31.900 It's rarely done these days. 00:26:31.900 --> 00:26:32.900 It's difficult to do. 00:26:32.900 --> 00:26:38.166 Well, there have been numerous, articles written about how the technical 00:26:38.900 --> 00:26:42.033 implementation is, somewhat challenging. 00:26:42.033 --> 00:26:45.033 And, reproducibility is low. 00:26:45.100 --> 00:26:49.200 Cd38 is also associated with for mutation status. 00:26:49.833 --> 00:26:53.333 Some studies suggest it may be an independent prognostic indicator. 00:26:55.166 --> 00:26:57.266 And it's commonly done in flow cytometry 00:26:57.266 --> 00:27:01.266 just because cd38 is included in most versatile tree panels. 00:27:02.533 --> 00:27:05.533 So it's already there, so to speak. 00:27:05.733 --> 00:27:10.433 You know, CD 49 D is, relatively new, marker 00:27:10.966 --> 00:27:14.200 that's been proposed for prognostic, testing. 00:27:15.366 --> 00:27:18.033 It's been shown to be an independent prognostic indicator 00:27:18.033 --> 00:27:21.100 and at least five studies that I know of so far, but 00:27:21.100 --> 00:27:24.100 it hasn't been included in current, guidelines. 00:27:26.966 --> 00:27:28.000 Molecular findings. 00:27:28.000 --> 00:27:31.200 So in addition to Tp53 mutation 00:27:31.200 --> 00:27:35.600 or Tp53 loss by, Sadr 00:27:35.600 --> 00:27:39.300 genetics or Fish, mutations in the Tp53 00:27:39.300 --> 00:27:42.300 locus are also considered bad, 00:27:42.666 --> 00:27:45.666 as are mutations in, atm 00:27:46.666 --> 00:27:49.033 3P1 notch 00:27:49.033 --> 00:27:52.033 signaling pathway mutations, particularly notch. 00:27:52.300 --> 00:27:55.033 And nowadays, these tests are all done 00:27:55.033 --> 00:27:58.033 as part of a single gene assay. 00:27:58.733 --> 00:28:02.733 Or sorry, nowadays these tests are all being done on NGS. 00:28:02.766 --> 00:28:05.366 Formerly they were single gene assays. 00:28:05.366 --> 00:28:07.933 They were done reflexively and took a long time. 00:28:07.933 --> 00:28:12.333 But you can do well over 40 genes in one go, on a next generation 00:28:12.333 --> 00:28:13.200 sequencing panel. 00:28:13.200 --> 00:28:16.233 And so people have really moved on to NGS. 00:28:17.433 --> 00:28:19.133 One biochemical 00:28:19.133 --> 00:28:22.133 prognostic factor is the beta two micro globulin. 00:28:23.266 --> 00:28:26.800 On the pathology side, we really don't see this because that's 00:28:28.066 --> 00:28:29.866 test sets ordered, 00:28:29.866 --> 00:28:34.000 from the clinical are from the, clinical floor and bypasses. 00:28:34.000 --> 00:28:36.900 And it's completely. 00:28:36.900 --> 00:28:39.533 So let's talk about minimal residual disease testing. 00:28:39.533 --> 00:28:42.533 Topic near and dear to my heart. 00:28:42.666 --> 00:28:44.200 In MRD, there's 00:28:44.200 --> 00:28:47.200 prognostic value and predictive value. 00:28:47.233 --> 00:28:48.666 What do these mean. 00:28:48.666 --> 00:28:50.133 Prognostic value means 00:28:50.133 --> 00:28:53.733 I know that you'll do poorly, but there's nothing I can do about it. 00:28:54.266 --> 00:28:57.266 Predictive tests or predictive value. 00:28:57.733 --> 00:29:00.333 Suggest or means I know that you're going to do 00:29:00.333 --> 00:29:04.366 poorly and I can treat you differently with good results. 00:29:05.700 --> 00:29:09.200 So really, the difference between prognostic and predictive values 00:29:09.200 --> 00:29:12.833 or predictive, tests is whether 00:29:12.833 --> 00:29:17.066 our clinical colleagues can actually use this information to affect outcomes. 00:29:18.166 --> 00:29:21.900 CLO minimal residual disease testing is certainly prognostic. 00:29:22.266 --> 00:29:25.866 The data data shows very clearly that CLL 00:29:27.133 --> 00:29:31.400 MRD will tell will separate out who's going to do well 00:29:31.400 --> 00:29:34.800 and who's going to do poorly regarding predictive value. 00:29:34.800 --> 00:29:36.600 Those studies are still ongoing. 00:29:36.600 --> 00:29:41.033 We don't really know with any certainty whether we can change 00:29:41.700 --> 00:29:45.500 the prognosis of patients with CLO, positive, 00:29:46.233 --> 00:29:49.233 minimal residual disease. 00:29:50.633 --> 00:29:52.900 In terms of minimal residual disease testing, 00:29:52.900 --> 00:29:55.900 there are two major modalities 00:29:55.966 --> 00:29:59.000 flow cytometry and next generation sequencing. 00:30:00.066 --> 00:30:03.733 Flow cytometry has sensitivities between 00:30:04.266 --> 00:30:06.900 .01 and .00 1%. 00:30:06.900 --> 00:30:10.566 So we're talking 4 to 5 log sensitivity. 00:30:11.600 --> 00:30:16.200 And what it looks for is, even a thin, typically aberrant population 00:30:16.400 --> 00:30:21.133 that stands out from normal B cell, background populations, 00:30:21.866 --> 00:30:26.200 it's widely applicable doesn't require you to know the phenotype 00:30:26.333 --> 00:30:30.500 a priori and has under 24 hour turnaround time. 00:30:30.766 --> 00:30:34.900 Flow cytometry is a very fast test and relatively cheap, all things considered. 00:30:36.733 --> 00:30:37.900 On the other hand, 00:30:37.900 --> 00:30:42.400 next generation sequencing, also called deep sequencing by 00:30:42.400 --> 00:30:46.700 some people, has the potential to give you six log sensitivity. 00:30:46.700 --> 00:30:51.266 So at least an order of magnitude greater sensitivity than flow cytometry. 00:30:52.233 --> 00:30:56.900 This looks for the same immunoglobulin sequence as the original tumor. And 00:30:56.900 --> 00:31:00.533 so really it's only applicable if you have the original tumor 00:31:00.533 --> 00:31:03.533 to go sequence. 00:31:03.566 --> 00:31:06.166 Purported turnaround time is 1 to 2 weeks. 00:31:06.166 --> 00:31:10.633 Optimistically, in reality, we see turnaround times of up 00:31:10.633 --> 00:31:13.933 to four weeks for these types of, next generation sequencing 00:31:13.933 --> 00:31:16.933 tests. 00:31:18.000 --> 00:31:20.500 That are up, we do CLIA 00:31:20.500 --> 00:31:25.233 by flow cytometry, using the Eric, consensus panel. 00:31:25.266 --> 00:31:28.533 This is called the European Research Initiative on CLL. 00:31:28.900 --> 00:31:34.333 It's a very well validated panel that describes, set of markers 00:31:34.333 --> 00:31:40.233 that, have been shown to be useful in, finding these small populations. 00:31:41.400 --> 00:31:44.633 This panel is actually platform and reagent independent. 00:31:44.666 --> 00:31:47.666 You can use this combination, 00:31:47.833 --> 00:31:51.333 either as an eight color, six color or ten color panel. 00:31:52.300 --> 00:31:55.433 And for the most part, if you validate it independently, 00:31:55.433 --> 00:32:00.400 you can get down to 0.00 1%, limited detection. 00:32:00.400 --> 00:32:01.933 So about five long. 00:32:01.933 --> 00:32:05.100 And that really is dependent on you collecting about 5 million cells 00:32:05.100 --> 00:32:06.300 for analysis. 00:32:06.300 --> 00:32:09.566 So in the peripheral blood that's a trivial process 00:32:09.566 --> 00:32:12.566 because you can collect as much peripheral blood as you want. 00:32:13.200 --> 00:32:15.866 But in the bone marrow that might be a little bit 00:32:15.866 --> 00:32:19.166 more challenging, especially in patients who've been treated and have, 00:32:20.100 --> 00:32:23.100 marrow hyperplasia or marrow fibrosis, 00:32:23.766 --> 00:32:27.700 but for the most part, clamped by flow cytometry is a peripheral 00:32:27.700 --> 00:32:30.700 blood based assay. 00:32:30.900 --> 00:32:34.266 On the contrary, by next generation sequencing, 00:32:34.566 --> 00:32:38.866 this really one only one commercial assay out there, to my knowledge, 00:32:39.133 --> 00:32:43.233 called the clonal CCK assay by adaptive, Biotechnologies. 00:32:44.133 --> 00:32:45.600 This requires, 00:32:45.600 --> 00:32:49.633 sent out to a centralized facility, I believe in Seattle, Washington. 00:32:49.633 --> 00:32:53.933 And as I said before, they support 1 to 2 week turnaround times. 00:32:53.933 --> 00:32:57.000 But really, people may see 1 to 4 week turnaround times. 00:32:57.733 --> 00:33:00.800 It requires our prior knowledge of the sequence, which is, 00:33:01.366 --> 00:33:03.833 a challenge if you don't know or if you can't 00:33:03.833 --> 00:33:08.433 get access to, original diagnostic material and the test costs 00:33:08.433 --> 00:33:11.633 $1,900 compared to ten color 00:33:11.633 --> 00:33:14.766 flow panel, which might cost about $280. 00:33:15.766 --> 00:33:17.866 Finally, this is a question 00:33:17.866 --> 00:33:21.033 that's still unanswered of whether the additional, 00:33:22.200 --> 00:33:25.033 sensitivity, the extra log sensitivity compared 00:33:25.033 --> 00:33:28.033 to flow cytometry really matters. 00:33:28.300 --> 00:33:31.166 As I said before, 00:33:31.166 --> 00:33:35.333 minimal residual disease and CL is prognostic but not yet predictive. 00:33:35.333 --> 00:33:37.033 And so, 00:33:37.033 --> 00:33:39.666 we know that lower and lower 00:33:39.666 --> 00:33:42.666 levels of MRD are correlated with better prognosis, 00:33:42.866 --> 00:33:46.166 but we don't know whether that lower and lower level of MRD, 00:33:47.166 --> 00:33:50.166 means that we can treat these patients differently. 00:33:52.766 --> 00:33:55.766 One of the major things in claimed 00:33:55.800 --> 00:33:59.400 research has been its use as a surrogate endpoint. 00:34:00.033 --> 00:34:03.900 So the issue is, survivals in CLO are measured in years. 00:34:04.333 --> 00:34:07.333 And so doing a clinical trial, 00:34:07.433 --> 00:34:12.166 where the endpoint is death or relapse, could be a challenge. 00:34:12.166 --> 00:34:16.900 You could be waiting years to get enough patients to die of disease, 00:34:17.233 --> 00:34:20.233 to be able to say whether your drug worked or not. 00:34:20.800 --> 00:34:23.400 So recently, in the past two years, 00:34:23.400 --> 00:34:28.233 the FDA has actually approved, Clomid as a surrogate endpoint. 00:34:28.666 --> 00:34:32.033 And so this is very important in drug trials that want to see whether 00:34:32.200 --> 00:34:33.466 their drug works, 00:34:34.433 --> 00:34:35.133 testing 00:34:35.133 --> 00:34:38.400 the minimal, minimal residual disease status in these patients, 00:34:39.033 --> 00:34:43.166 gives them a good indication as to whether these patients are going to do 00:34:44.033 --> 00:34:47.033 well or do poorly. 00:34:49.200 --> 00:34:49.800 Targeted 00:34:49.800 --> 00:34:52.833 therapies are also a big thing in CLO. 00:34:53.266 --> 00:34:55.900 They're a big thing in, numerous neoplasia. 00:34:55.900 --> 00:35:00.366 But, CLL has been kind of the poster child for targeted therapies. 00:35:00.933 --> 00:35:05.933 So anti CD 20, so-called toxin map, although there are other 00:35:05.933 --> 00:35:10.833 generic formulations nowadays, has been around for 20 years now. 00:35:11.833 --> 00:35:16.100 And it's really the backbone of any B-cell, therapy. 00:35:18.800 --> 00:35:20.200 FCR flu there 00:35:20.200 --> 00:35:24.433 being cyclophosphamide and rituximab is a common therapy for CLL, 00:35:25.033 --> 00:35:30.066 chloride, bison, chlorine B-cells, and rituximab, is typically used 00:35:30.066 --> 00:35:33.600 in patients who can't, tolerate the cyclophosphamide and 00:35:34.100 --> 00:35:36.600 are Darby, 00:35:36.600 --> 00:35:39.500 we joke in pathology that they just use for toxin. 00:35:39.500 --> 00:35:42.500 We, they they put some AB in the water. 00:35:43.100 --> 00:35:45.766 It's so commonly used for based on the ablations. 00:35:46.733 --> 00:35:49.100 So that's kind of the old school 00:35:49.100 --> 00:35:53.866 treatment, 15, 20 years newer, targeted therapies 00:35:53.866 --> 00:35:57.966 include Burton, tyrosine kinase inhibitors, BTK inhibitors. 00:35:58.366 --> 00:36:01.366 And these have, 00:36:01.400 --> 00:36:03.366 use even in high risk patients. 00:36:03.366 --> 00:36:07.300 So patients who have Tp53 mutations, complex 00:36:08.033 --> 00:36:11.033 carrier types, these 00:36:11.033 --> 00:36:14.033 these target the BTK, pathway. 00:36:14.666 --> 00:36:18.266 And they have a characteristic name, ending in Bruton ed, 00:36:18.833 --> 00:36:21.633 as opposed to the Rotax A Mab, the Mab 00:36:21.633 --> 00:36:24.633 standing for monoclonal antibody. 00:36:26.266 --> 00:36:30.033 PI3 kinase inhibitors, are coming onto the market. 00:36:30.333 --> 00:36:33.333 The prototypical one here is ideal. 00:36:33.433 --> 00:36:36.300 It's being increasingly used, but, 00:36:36.300 --> 00:36:39.333 nowhere near the numbers of, BTK inhibitors. 00:36:39.933 --> 00:36:44.566 And then finally BCL two inhibitors, are being widely 00:36:44.566 --> 00:36:50.500 used in a variety of neoplasia, including AML, straight and MDS. 00:36:50.733 --> 00:36:53.733 So, opposite of lymphoid malignancies. 00:36:53.733 --> 00:36:56.433 They're being used in myeloid malignancies as well. 00:36:56.433 --> 00:37:00.900 But BCL two inhibitors have a distinctive name ending in clacks. 00:37:00.900 --> 00:37:03.133 So the prototypical one is venetoclax. 00:37:05.300 --> 00:37:08.300 It turns out that many B-cell lymphomas, 00:37:09.300 --> 00:37:11.566 are considered addicted 00:37:11.566 --> 00:37:15.800 to BCL two, which is also called a B-cell lymphoma. Two 00:37:16.800 --> 00:37:20.333 and these this is an anti apoptotic protein 00:37:20.800 --> 00:37:26.433 that when upregulated or overexpressed leads to cell immortality. 00:37:26.900 --> 00:37:31.366 So you can see how you have having immortal cells 00:37:31.733 --> 00:37:34.733 that never kill themselves off can lead to neoplasia. 00:37:37.700 --> 00:37:40.900 Now why do I go into this tangent about target therapies? 00:37:41.266 --> 00:37:44.300 Because there are important implications in the clinical lab. 00:37:45.233 --> 00:37:47.533 Loss of targets. 00:37:47.533 --> 00:37:49.766 So patients who get anti 00:37:49.766 --> 00:37:55.700 CD 20 or antecedent 19 therapies, you'll oftentimes see a loss of the target 00:37:55.700 --> 00:37:59.900 for detection in minimum in minimal residual disease testing. 00:38:01.066 --> 00:38:03.133 This could be a problem for us 00:38:03.133 --> 00:38:07.166 because we can falsely call patients negative for disease. 00:38:07.666 --> 00:38:11.666 Mainly because we don't have a marker to detect disease in these treated patients. 00:38:12.266 --> 00:38:14.166 But what do you do in this situation? 00:38:14.166 --> 00:38:16.833 First of all, you do what's necessary to treat the patient. 00:38:16.833 --> 00:38:20.833 So these patients are going to get targeted therapies no matter what. 00:38:21.466 --> 00:38:24.500 No matter what the implications are for residual disease testing. 00:38:25.200 --> 00:38:28.600 The other thing is to look for alternative markers of residual disease. 00:38:28.933 --> 00:38:32.866 So even if CD 20 is knocked out or CD 19 is knocked out, 00:38:33.300 --> 00:38:36.666 additional B-cell markers like CD 22 could be used. 00:38:37.400 --> 00:38:39.766 CD 24 is an emerging B-cell marker 00:38:39.766 --> 00:38:44.400 that could be useful in, in heavily pretreated patients. 00:38:45.400 --> 00:38:46.500 But in 00:38:46.500 --> 00:38:49.533 the at the end of the day, we have to do what's best for the patient. 00:38:50.300 --> 00:38:53.533 And on the lab side, we just need to deal with it 00:38:53.533 --> 00:38:58.566 as best as we can, using less ideal markers that still work. 00:38:59.333 --> 00:39:03.300 The other implication for targeted therapies is, an increasing, 00:39:04.000 --> 00:39:08.033 number of requests to test for a target for therapeutic purposes. 00:39:08.600 --> 00:39:13.133 So I, it's not infrequent for me to get, questions about, 00:39:13.400 --> 00:39:18.633 oh, what's the level of Cd19 expression on this, B-cell neoplasm or what's level 00:39:18.633 --> 00:39:21.766 city 20 expression on this B-cell neoplasm? 00:39:22.533 --> 00:39:26.400 With the idea of using, targeted therapies. 00:39:27.000 --> 00:39:30.833 The issue is that this needs to be done at the time of diagnosis 00:39:31.133 --> 00:39:34.133 or at the time of relapse. 00:39:34.166 --> 00:39:37.166 It's very difficult to do, 00:39:38.000 --> 00:39:41.033 this type of testing on, MRD 00:39:41.133 --> 00:39:44.266 specimens, mainly because you don't have a lot of tumor. 00:39:44.833 --> 00:39:47.433 And we know that there's significant tumor heterogeneity. 00:39:47.433 --> 00:39:52.700 And so if you're not sampling enough cells, you could be falsely saying 00:39:52.700 --> 00:39:56.066 that the tumor is positive or negative for this marker. 00:39:56.066 --> 00:39:59.633 And they're thereby either wasting a marker on a patient 00:39:59.633 --> 00:40:03.500 who doesn't have it or, sorry, wasting a targeted therapy on a patient 00:40:03.500 --> 00:40:07.333 who doesn't have this disease, this disease marker or, 00:40:08.800 --> 00:40:10.566 denying them this targeted 00:40:10.566 --> 00:40:13.566 therapy when they could benefit from it. 00:40:15.233 --> 00:40:16.866 So with that, 00:40:16.866 --> 00:40:19.866 CLL is a common and usually indolent disease. 00:40:20.533 --> 00:40:24.900 Flow cytometry is considered the fastest and cheapest way to make this diagnosis. 00:40:25.500 --> 00:40:28.966 It's, clinically important to really separate out indolent 00:40:28.966 --> 00:40:31.100 from aggressive disease. 00:40:31.100 --> 00:40:33.800 That is to call it prognostication, 00:40:33.800 --> 00:40:38.466 because these patients will be treated differently. 00:40:38.666 --> 00:40:41.966 Whether or not we can change outcomes is still an open question, 00:40:42.133 --> 00:40:45.133 but they are treated differently because, 00:40:45.766 --> 00:40:47.700 it would be a shame 00:40:47.700 --> 00:40:51.333 to let a patient, who has indolent disease 00:40:51.333 --> 00:40:54.333 go through more treatment than they need to. 00:40:54.633 --> 00:40:57.633 Prognostication is is based on, 00:40:57.933 --> 00:41:00.966 grading so that, the morphologic, 00:41:01.966 --> 00:41:04.966 features of the disease staging, the, 00:41:05.100 --> 00:41:10.033 anatomic distribution of disease, molecular mutations, side 00:41:10.033 --> 00:41:13.033 or genetic abnormalities, 00:41:14.066 --> 00:41:16.433 and so on and so forth. 00:41:16.433 --> 00:41:19.500 And with that, conclude my talk and thank you. 00:41:19.500 --> 00:41:21.400 And open up for any questions 00:41:22.966 --> 00:41:26.500 I do see, one submitted in the chat is 00:41:26.766 --> 00:41:30.433 do you see increased incidence of CML 00:41:30.433 --> 00:41:34.233 with rheumatoid arthritis, drug therapies or other treatments? 00:41:36.633 --> 00:41:38.100 Personally, I do not. 00:41:38.100 --> 00:41:41.266 There has been, theoretical possibility, 00:41:41.266 --> 00:41:44.266 but I don't think it's been proven. 00:41:44.900 --> 00:41:47.900 Typically with these types of, 00:41:49.500 --> 00:41:51.866 autoimmune drugs, 00:41:51.866 --> 00:41:56.733 they are sometimes cytotoxic and can lead to other types of diseases. 00:41:56.733 --> 00:42:00.433 Myeloid disorders are more common. So, 00:42:01.600 --> 00:42:05.366 methotrexate, for example, has been associated with MDS and things like that. 00:42:06.166 --> 00:42:10.100 But classical really hasn't had any drug reported 00:42:10.200 --> 00:42:13.533 or drug, associations, reported.