Discovering A Monoblast Marker
April 08, 2022- 00:00Today's grand round speaker
- 00:02is Mina Shu or very own Mina.
- 00:06She needs no introduction.
- 00:09Actually, Mina has been here longer than I,
- 00:12but I'll introduce Mina for the
- 00:15benefit of the new folks Mina grew up
- 00:20in California and came to northeast
- 00:23for college at Harvard University,
- 00:27and then she went back for MD at.
- 00:30UCSF and she must have missed
- 00:34the seasons and the snow,
- 00:36so she came back to Yale
- 00:39to train in pathology.
- 00:41During her training,
- 00:42Mina rose to become chief resident and Mina
- 00:47showed an early interest in hematology.
- 00:50She spent a year doing research
- 00:54at Brigham and Women's Hospital.
- 00:59During her medical school and then after
- 01:03finishing her pathology residency at Yale,
- 01:07she went back to Brigham and Women's
- 01:11Hospital to do a clinical fellowship.
- 01:14And as minard's,
- 01:16if you logged in earlier you
- 01:19heard me say there was no room
- 01:22to do anything else but clinical
- 01:25work with voice extremely heavy.
- 01:30Fortuitously, you know was recruited
- 01:33right back at faculty position at
- 01:36Yale pathology, and we are very
- 01:39lucky to have Meena here with us.
- 01:43Mina has established a track record of
- 01:47pursuit of curiosity and excellence in
- 01:50all her endeavors and I would say all her
- 01:54endeavors because she won many grants,
- 01:57awards and scholarships.
- 01:59From early age, in fact,
- 02:02several in high school making it
- 02:05to Harvard University Dean's list.
- 02:09At Yale, an outstanding achievement
- 02:12in autopsy or world during residency.
- 02:15Several Chairmans challenge grant
- 02:18awards and a Gilleard foundation
- 02:21grant that is just to name a few.
- 02:25Mina is a consummate clinician and
- 02:28an excellent research collaborator.
- 02:31She has close to add publications
- 02:34to her credit. In addition,
- 02:37Mina is an outstanding teacher and mentor,
- 02:40and that's demonstrated by her various
- 02:44speaking engagements all over US,
- 02:47Canada, and also China.
- 02:49She has given short courses,
- 02:52interactive microscopy sessions and
- 02:55participated in workshops and symposia
- 02:59at annual meetings of the US and
- 03:03Canadian Academy of the Theology.
- 03:06And at other professional organizations.
- 03:09And her mentees,
- 03:11mostly residents and fellows,
- 03:13they have successfully presented
- 03:16abstracts at national and international
- 03:19meetings and published the manuscripts
- 03:22in impactful journals.
- 03:24So with no further ado,
- 03:27let me now present her work today.
- 03:31You know?
- 03:36Thank you so much mandjou for
- 03:39that really amazing introduction
- 03:42that I probably don't deserve,
- 03:45but I just wanted to say, you know,
- 03:47thank you also for asking me to do
- 03:50this talk when you first asked me,
- 03:52I wasn't sure which of the many
- 03:55cool stories that were involved
- 03:57in in heme path I should present,
- 03:59but I quickly decided to do this one
- 04:02because I think you would agree with me.
- 04:05And and probably malani as
- 04:08the current IHC director.
- 04:10That now is a good time to tell
- 04:13our trainees and students about the
- 04:15importance of discovering new markers
- 04:18and that we are not at the end of the
- 04:21IHC era but at the beginning and we
- 04:25will get even better during the next ten,
- 04:2920-30 years at investigating protein
- 04:32expression in human tissues.
- 04:34And then cancer in particular.
- 04:37So these are my COI disclosures.
- 04:40I don't think that any of this
- 04:42will impact our talk today.
- 04:44And this is my outline,
- 04:45so I wanted to do a case presentation of a
- 04:50patient recently seen this phantom menace,
- 04:53which is our counting of blast and
- 04:56blast equivalents on heme path.
- 04:58A New Hope or marker for AMOL,
- 05:01and then a few other projects
- 05:04I spun off the original one.
- 05:07And by the way,
- 05:08I am not actually a Star Wars geek.
- 05:11Some of my friends are and they.
- 05:15I have thought in their childhood
- 05:17that becoming a Jedi is a legitimate
- 05:20career choice,
- 05:21which I think is kind of funny
- 05:23because as a pathologist,
- 05:24sometimes I think we are living
- 05:27that dream because we're actually
- 05:30able to on a daily basis,
- 05:32force miniscule pieces of tissue
- 05:35to tell us their truth and that to
- 05:39me is amazing and quite Jedi like.
- 05:41So here's my books case presentation.
- 05:45This is a 58 year old woman with
- 05:47a 2 year history of chronic
- 05:50myelomonocytic leukemia,
- 05:51and I actually saw that initial CML
- 05:55diagnosis now with progressive cytopenias
- 05:57bone pain and circulating glass and
- 06:00she is admitted for the symptoms.
- 06:04So her peripheral blood showed 8% blasts.
- 06:07These are mono blastic cells.
- 06:12She also had degranulate poesis.
- 06:15So dysplasia of the granulocyte
- 06:17lineage with abnormal folding
- 06:19of the granulocytes and in other
- 06:21areas hypo granularity.
- 06:23She also had a monocytosis as is
- 06:25normally seen for her blood smear.
- 06:28These delicately folding cells
- 06:30with Gray blue cytoplasm.
- 06:32These are mature monos.
- 06:34They proceeded to do a
- 06:36bone marrow aspiration,
- 06:38but it was a dry tap,
- 06:39most likely because of the fibrosis
- 06:42in the core biopsy.
- 06:44This was the core biopsy in which you
- 06:46see it is hypercellular for her age and
- 06:49there is dysplasia in the megakaryocytes.
- 06:52You can also see a myeloid predominance
- 06:55with hardly any erythroid islands here.
- 06:59But at least there is maturation in the
- 07:02form of metamyelocytes granulocytes.
- 07:04So actually if you compare to
- 07:07her initial bone marrow biopsy,
- 07:09this area looks very similar
- 07:11to the initial one,
- 07:13but a little more hypercellular.
- 07:15However,
- 07:16about 30% of the bone marrow
- 07:19actually showed these
- 07:20foci of immaturity.
- 07:22So here I say immaturity because the
- 07:24cells have more dispersed chromatin,
- 07:27some distinct nucleoli.
- 07:29And you're having a lack
- 07:31of the mature granulocytes,
- 07:34so this is very worrisome,
- 07:35especially in this clinical context
- 07:39of transformation into AML.
- 07:42So can we treat this patient as AML now?
- 07:45Well, the definition of AML is
- 07:4820% loss in blood or bone marrow.
- 07:50So how can we reach that with
- 07:52our without our gold standard
- 07:53of the aspirin count?
- 07:57If the AML expressed our most
- 07:59utilized immunostain which is CD 34,
- 08:02we could demonstrate that on core biopsy,
- 08:05but without that marker.
- 08:06It would be very difficult to substantiate
- 08:09the cytologic blast count on core,
- 08:12and this was actually negative,
- 08:14as we knew from before.
- 08:17So just taking a step back and L is a
- 08:20genetically heterogeneous myeloid neoplasm,
- 08:23and while the FAAB classification is
- 08:25not employed in clinical use now,
- 08:28it is still the most adherent to
- 08:31myeloid differentiation status,
- 08:32so you'll see that it still comes into
- 08:34play in current research studies.
- 08:37The overall poor 5 year old survival
- 08:39for AML as well as the relapse
- 08:41rate continue to be a huge problem.
- 08:46You can see that some improvements
- 08:48have been made in the last couple
- 08:50of decades as seen here in Black is
- 08:542000 to 2006 and yellow is until
- 08:572011 and blue is the most current.
- 09:00This is from a Danish study that I'm
- 09:02using because it's one of the most recent,
- 09:04but those published from the
- 09:06US show similar statistics.
- 09:08You can see that there is still a long
- 09:10way to go for patients over the age of 60.
- 09:16In broad strokes, some of the
- 09:18major AML subtypes are classified
- 09:20according to cytogenetic findings
- 09:22that include balanced translocations.
- 09:25Molecular findings also
- 09:27help in risk stratification.
- 09:29Older age remains one of the most
- 09:31major risk factors for poor outcome.
- 09:36The foundation of treatment for
- 09:38new AML is induction chemotherapy
- 09:41followed by either consolidation,
- 09:43chemo or consideration toward
- 09:45allogeneic stem cell transplant.
- 09:48Newer therapeutic options include
- 09:50small molecule inhibitors, and in 2018,
- 09:53BCL 2 inhibitor venetoclax was
- 09:55approved as a combo regimen,
- 09:57typically with hypomethylating agents,
- 09:59such as a deciding that does
- 10:02show great effect in older AML
- 10:04patients though durable remission.
- 10:06Is still difficult to obtain.
- 10:09So going back to our case,
- 10:11the problem is whether we can
- 10:13diagnose AML in this particular
- 10:15biopsy where you have some areas
- 10:17of maturation and some not,
- 10:19and a absent aspirate,
- 10:22not just bad aspirate and just to show you
- 10:26in cases where you do get a good asper smear.
- 10:29This is the gold standard.
- 10:30The gold standard is counting of 500
- 10:32cells in each patient to enumerate
- 10:35not just myeloblasts and monoblast,
- 10:37but in the case of monocytic leukemias.
- 10:39Pro monocytes,
- 10:40which are considered blast equivalents
- 10:43but not to include mature monocytes.
- 10:46So in this study in which they had 14
- 10:50hematopathologist do consensus counting
- 10:51of a number of cases on blood and aspirate.
- 10:55These two are mono blasts so clearly blasts.
- 10:59This one is the pro monocyte.
- 11:02So just a step towards maturation,
- 11:05but still a blast equivalent.
- 11:07And here is a mature monocyte.
- 11:10And of course they use the
- 11:12best picture presentation.
- 11:13This is in a peripheral blood and
- 11:15just to make the situation worse,
- 11:18CML is further stratified.
- 11:20So this is the chronic counterpart to AML
- 11:24is stratified further by this last county.
- 11:29And concordance can be very difficult
- 11:31to achieve even among these expert
- 11:34hematopathologist only getting 2
- 11:36consensus at 74% which just to be clear,
- 11:40I don't think is good concordance.
- 11:43When you're trying to get a
- 11:45patient into chemo for AML.
- 11:47So the challenge is that even
- 11:49in the best possible scenario,
- 11:51like an excellent aspirant,
- 11:52the counting of Mono City lost equivalents
- 11:55is riddled with reliability issues,
- 11:58good as spirits are harder
- 11:59and harder to come by.
- 12:01This is something that when I
- 12:03talk to senior hematologist they
- 12:06really feel in their bones that
- 12:08it has become like a lost art,
- 12:10very difficult to get good aspirates.
- 12:12I'm on service this week and Cohen and I
- 12:15were guessing that about three of our 20.
- 12:17Last day or so we're good aspirates.
- 12:22Some of it is due to treatment.
- 12:24The newer treatments can lead to fibrosis.
- 12:26There are procedural issues.
- 12:29There's going towards IR which may not be.
- 12:32You know,
- 12:33as invested in looking at the aspirates
- 12:36later and there is no reliable
- 12:38monoblast marker on biopsy material.
- 12:40So just to keep in mind that aspirate
- 12:43where that material comes from is
- 12:45the same tube that the flow and.
- 12:48Heterogenetic S are deriving
- 12:49their specimen from so it kind of
- 12:52hurts us on multiple levels,
- 12:53but the core biopsy remains good.
- 12:55The core biopsy is still coming from
- 12:57that same jump shooting needle and
- 12:59it can add additional information
- 13:02beyond the aspirate in terms of
- 13:05architecture and localization.
- 13:07This is what I like to call
- 13:09the tree of myeloid life.
- 13:10So we start here with the
- 13:13hematopoietic stem cell going towards
- 13:15myeloid progenitors that then go
- 13:17toward GMP becoming granulocytes
- 13:20or monos and dendritic cells.
- 13:24So just in case you're wondering
- 13:26about mono markers in general,
- 13:29we do have a lot of sorry.
- 13:31A lot of immunostains and flow
- 13:34markers like these that will mark.
- 13:37All of these mono lineage cells,
- 13:42but they do not differentiate
- 13:45between blast versus mature.
- 13:47On the other hand,
- 13:48CD 34 is a great marker for glass
- 13:51that are positive for it,
- 13:53so the granular acidic glass are positive
- 13:55for CD34 whereas the granulocytes are not.
- 13:59So what we're looking for is something here.
- 14:05OK. Is there something with the sound
- 14:09ohh you're good now,
- 14:11OK? So we went on the hunt for.
- 14:15A phenotype. That is strongly
- 14:18strongly expressed by mono precursors,
- 14:21but not expressed in later stages,
- 14:24and in this older study using human
- 14:27umbilical cord blood and bone marrow,
- 14:29they were able to fractionate
- 14:32GMP into four subpopulations.
- 14:35Human common monocyte progenitors are one
- 14:38of the subpopulations here that do not
- 14:42show any potential for differentiating
- 14:44into myeloid or lymphoid cells,
- 14:46and according to.
- 14:48Gene expression profiling I of eight
- 14:50here seems to show the features that
- 14:53we're looking for in terms of being
- 14:56expressed in early mono progenitors,
- 14:58but not in their later stages.
- 15:01We also considered an R481,
- 15:04but didn't have as much supporting
- 15:07data in the literature.
- 15:09Fire Eight was also a great
- 15:10candidate for us because there was
- 15:13a commercially available antibody
- 15:15for purchase and testing.
- 15:16Some of you might say to yourself,
- 15:18wait, I just heard about IRV for some
- 15:22reason and you did last week when
- 15:25we had Lee Grimes from Cincinnati.
- 15:27Come and talk to us about his work
- 15:30in severe congenital neutropenia.
- 15:31He actually used IRF 8 in his recent
- 15:35studies as a negative control in
- 15:38the balance between granulocyte
- 15:40and monocyte differentiation.
- 15:42If I that paper actually came out later
- 15:44than when we proceeded down this pathway.
- 15:47But if we had seen it at that time,
- 15:49I think it would have given us further
- 15:51support to pursue this marker.
- 15:55So RV is a master transcriptional
- 15:58regulator of monocyte development,
- 15:59and it regulates monocyte differentiation
- 16:01genes that we know about is strongly
- 16:04induced by interferon gamma in
- 16:06the setting of infection and its
- 16:08first expressed after that comment.
- 16:10Granulocyte monocyte progenitor stage.
- 16:12Its expression is maintained at much lower
- 16:16levels in monos Max and dendritic cells,
- 16:18but not in neutrophils.
- 16:20It promotes apoptosis via activation
- 16:23of facts and repression of.
- 16:25CL2 and ECL Excel and loss of IRA in mice
- 16:29leads to an expansion of granulocytes,
- 16:32decreased monos,
- 16:33decreased's and a CML like picture and
- 16:37in fact overexpression of IR of eight
- 16:39inhibits BCR 8 ball driven leukemogenesis.
- 16:42It's transcripts are greatly reduced
- 16:44and CML patients and it's so far acts
- 16:48as a tumor suppressor in mouse a PML.
- 16:50So you might wonder what prompted me
- 16:53to look at this marker in an acute.
- 16:56Leukemia and I have to say at that time.
- 17:00I just really want to test it out.
- 17:01Given the earlier gene expression data,
- 17:05even though there was not much known
- 17:08about it acting as an oncogene in AML,
- 17:11and in fact it was the reverse,
- 17:13but later studies did show that
- 17:15it was a good hunch.
- 17:18So we started doing this validation
- 17:21on a cornucopia of tissues.
- 17:24This is susmita adapala who actually
- 17:26had done Hurricane Path Fellowship
- 17:28before she came to Yale to be a
- 17:31research fellow with us for a year,
- 17:33and she is now a hematopathologist at LJ,
- 17:36so she did.
- 17:38The scouring of literature for this
- 17:40gene expression profile and helped
- 17:42me get started on this validation.
- 17:45You can see that our of eight.
- 17:47Does in fact stain B cells in
- 17:50follicles in their mantle zone
- 17:51and in the germinal center.
- 17:53This is myeloid sarcoma.
- 17:55In soft tissue it stains the tumor cells
- 18:00and it is negative in this carcinoma,
- 18:04but you can see in the background
- 18:06stroma here that there are granular
- 18:08sites and there are histiocytes and
- 18:11those did not stand for our marker.
- 18:13So then I went ahead and pulled some of
- 18:16our decalcified core bone marrow because
- 18:19we want to use this on decalcified tissue.
- 18:23This is what we get most often
- 18:25and here the blast counting is by
- 18:28the aspirate or gold standard.
- 18:30So here is an initial diagnosis.
- 18:32Am OL monocytic leukemia at
- 18:34more than 90% blast.
- 18:36Here is a normal staging bone
- 18:38marrow for Hodgkin lymphoma that
- 18:40was not involved and a residual.
- 18:43Disease or residual for AML at 10%
- 18:47loss in the aspirate and here is
- 18:491 at morphologic remission defined
- 18:51by less than 5% less.
- 18:54So then we pulled our whole
- 18:57cohort of 90 am OL.
- 18:59That included remission residual somewhere
- 19:01in between and also a smaller cohort
- 19:06of chronic myelomonocytic leukemia.
- 19:08Other AML.
- 19:10So ammo's not monocytic and normal for the
- 19:15normal control bone marrows we enriched.
- 19:18For the ones that had monocytosis in
- 19:21the peripheral blood from 10 to 30%.
- 19:24And you can see that. This.
- 19:30The different diagnosis they
- 19:31actually had the CBC,
- 19:33a presentation, treatment protocols
- 19:35and outcomes that were compatible
- 19:37with what you would expect to
- 19:39find for each disease category.
- 19:44The NGS showed that the tumors
- 19:47had typical molecular features,
- 19:49about half the leukemias had NPM 1 mutations
- 19:53and close to a third had FLIT 3 ITD.
- 19:56You can also see that for
- 19:59the monocytic leukemias,
- 20:00whether they're chronic or acute,
- 20:03that they were enriched for SRSF,
- 20:052 pathogenic variants,
- 20:06and of course, tattoo.
- 20:11Two practicing hematopathologist counted
- 20:13the stain on core biopsies and these
- 20:16are plotted here for each biopsy with
- 20:19correlation to their aspirate blast counts.
- 20:22Sam Katz really gets all the credit
- 20:24here for prompting me to do this
- 20:27project in the 1st place because
- 20:28if you don't know him well by now,
- 20:31he is a very eloquent complainer.
- 20:34And so while some people might just say,
- 20:37oh, I don't like to count 500 cells
- 20:39or this aspirin is really bad.
- 20:41He really hones down on the problem here
- 20:45and compelled me to go upon this search.
- 20:50So as you might know about what they say
- 20:53about good deeds not going unpunished,
- 20:56he had to be roped into the
- 20:58validation as well.
- 20:59So this was done independently
- 21:01with disregard for their diagnosis,
- 21:04and we achieved a pretty good correlation.
- 21:08This was the diagnostic test
- 21:11characteristics using aspera count
- 21:13as the surrogate for disease status,
- 21:16so AML being 20% plus or higher
- 21:19residual disease being 5% plus or
- 21:22higher and negative or residual being
- 21:25less than 5% as compared to IR 8 IHC
- 21:30result due to a reviewer question.
- 21:33We actually went back and did the
- 21:35same with CD 34 because we actually
- 21:38didn't know how well we're doing.
- 21:40CD34,
- 21:40as opposed to our aspirate blast
- 21:42count in the granulocytic leukemia
- 21:44and we did not actually get to
- 21:47the same good correlation.
- 21:48It was still good,
- 21:49but it was not quite at .8
- 21:51which we had for IR 8.
- 21:56And this is the correlation for CML,
- 21:59which is not as strong.
- 22:00But we also had a smaller cohort for CML.
- 22:04One of the reasons that CML cases might
- 22:08be especially difficult I believe,
- 22:11is that occasionally,
- 22:12as with our first case that I showed there
- 22:15is focal elevation of glass which may
- 22:18not be well represented on aspirin smear,
- 22:21because, as you might know,
- 22:24for aspirus you're really kind of sucking it.
- 22:26Thought from one specific point.
- 22:28So here is an interesting biopsy.
- 22:30We had a few years ago where you can
- 22:32see even on low power that this corner
- 22:35here looks different from this part.
- 22:37So most of the bone marrow showed
- 22:40as in the upper part maturing
- 22:42trilineage amount of polices.
- 22:44Some increase in boss because
- 22:46this person also had CML.
- 22:48That was a little bit elevated
- 22:50like CML one or CM L2,
- 22:52but then the lower right hand corner
- 22:54actually had a sheet of glass.
- 22:56As represented in E here and when
- 22:59we did our of eight you can see
- 23:01how dramatic this transition is.
- 23:03Almost like a solid tumor malignancy.
- 23:09I also get asked whether the
- 23:12macrophages are positive,
- 23:13and I think not.
- 23:15This is a AML that is not
- 23:18monocytic with a federal flage.
- 23:21Here this is a monocytic leukemia
- 23:23at initial diagnosis and this is
- 23:25one of our staging bone marrow
- 23:27biopsies showing a Cidra page here
- 23:29and they were negative for a marker.
- 23:33There were discrepancies in
- 23:35assessing residual disease,
- 23:37so 10 cases showed a discrepancy defined
- 23:41at where aspirate had less than 5% loss.
- 23:44But IRA expression was
- 23:46just a little above 5%,
- 23:49so three of these actually had
- 23:51definitive evidence of disease by
- 23:54cytogenetics as unbalanced translocations
- 23:56flit 3 ITD or MPM 1 mutations,
- 24:00and three have clinical relapse
- 24:01within two months of the biopsy.
- 24:04So I just want to point out here
- 24:06that our whole correlation our
- 24:07ground truth in this study has
- 24:09been the aspirate blast count.
- 24:11But that as we know,
- 24:13as pathologist is just another sample.
- 24:15It's a different sample from the core biopsy.
- 24:17So what is really the truth?
- 24:20Maybe it is the clinical behavior.
- 24:22Maybe it is genetics or molecular and
- 24:24and I think that that correlation comes
- 24:27into play when we're looking at a new marker.
- 24:30One had flow elevation of
- 24:32human tacones above 10%.
- 24:33Which pumped in me to go on a search for
- 24:35all the biopsies that had increased humidity.
- 24:38Phones, hard to find,
- 24:40but they're they are.
- 24:41IRA dusting hematogenous,
- 24:42so that could be a pitfall.
- 24:45In the rare case where you had
- 24:48really significant elevation by
- 24:50flow and then we had two remaining
- 24:52discrepancies of overcount by IR.
- 24:54RF eight as compared to aspirate
- 24:56at a low percentage blast,
- 24:59one did show RA expression that was
- 25:01lower than the aspire blast count.
- 25:03But upon evaluation,
- 25:05the core biopsy showed
- 25:07substantial aspiration artifact,
- 25:09so perhaps it should have been
- 25:11excluded in the first place.
- 25:12So, excluding the above cases,
- 25:14where disease was still present,
- 25:15the overall discrepancy was 3% with
- 25:19regard to residual disease assessment.
- 25:22So in conclusion,
- 25:23in Almal there is high correlation of IRV
- 25:26positive cells to aspirate last count.
- 25:29The comparison of IRV staining to
- 25:32blast count and see mammal also
- 25:33showed a pretty good correlation,
- 25:35though that requires more study and
- 25:38in contrast reactive monocytosis and
- 25:41AML without monocytic differentiation
- 25:43did not show IRA elevation when it was
- 25:47used to categorize cases as acute leukemia,
- 25:49positive or residual leukemia or negative.
- 25:52Sensitivity and specificity
- 25:53was high and this marker can be
- 25:56clinically useful as IHC to possibly
- 25:59diagnose and track disease.
- 26:01Particularly in cases of poor
- 26:04aspiration or focal blast increase.
- 26:07So shortly after this manuscript
- 26:09was accepted for publication,
- 26:11actually a great study came out
- 26:13in molecular cell that actually
- 26:15really helped me understand
- 26:16what we were seeing better.
- 26:18This is a summary of transcription
- 26:20factor domain focus.
- 26:21CRISPER screens genes were ranked
- 26:24by AML biased essentiality scores
- 26:26defined by the difference in
- 26:29a particular domain score in
- 26:31AML versus non AML cell lines.
- 26:33And I should point out that
- 26:35in the cell lines
- 26:36that showed. Particularly high IRF
- 26:398 dependency, those were monocytic,
- 26:42leukemias, and the others are not.
- 26:48Umm? Competition based proliferation
- 26:51assays performed in Castine positive
- 26:54cell lines show that AML cells
- 26:57that were transduced with IRF 8
- 26:59guided RNA were rapidly depleted
- 27:02and outcompeted by parental cells.
- 27:05In separate studies showing
- 27:09173 AML patient samples,
- 27:10High R of eight expression was seen in
- 27:14association with diverse cytogenetic
- 27:16and molecular driver mutations,
- 27:18so there's genetic and molecular features
- 27:20actually did not define this behavior.
- 27:28They also found that RFA is
- 27:29enriched at the MEF 2D locus.
- 27:31That is another transcription factor,
- 27:34and it is known to be important in
- 27:38some Bal in several cell lines,
- 27:41such as month 13,
- 27:42which is a monocytic cell line,
- 27:44RNA seek of gene expression.
- 27:45Changes indicate that cell
- 27:47lines transduced with guided
- 27:49RNA to IF8 revealed MEF 2D to
- 27:52be significantly downregulated.
- 27:56So transcription factors are
- 27:59difficult to therapeutically target,
- 28:01so they looked for a chromatin
- 28:03regulator upstream that would be
- 28:05more druggable in a separate crisper
- 28:07dropout screen that was focused
- 28:08on chromatin regulatory domains.
- 28:10They uncover CMU and eight as AML specific,
- 28:14and here you can see that in certain
- 28:17cell lines they are hypersensitive to
- 28:20depletion of this chromatin reader,
- 28:22and others are less so.
- 28:25The ones that are non AML are
- 28:27insensitive to it and by the way,
- 28:30cmda is ubiquitously expressed
- 28:31in all cell lines,
- 28:33so it was not because of how
- 28:35much the YD ate there was.
- 28:39They further found that all the
- 28:41AML cell lines are IR 8 high are
- 28:44hypersensitive to depletion of CMY D8
- 28:47and loss of CMD 8 resulted in decrease
- 28:50in ire of eight as well as Mick
- 28:52over time and an increase in Miller
- 28:55differentiation associated genes.
- 28:57Genetic depletion CMND in normal
- 28:59hematopoietic stem cells did
- 29:00not impact cells in vitro.
- 29:02So just what you want for a druggable target.
- 29:10Altogether and with other data that I'm
- 29:11not presenting in the interest of time,
- 29:13they show that IR 8 helps form
- 29:16a transcriptional circuit to
- 29:18support AML proliferation.
- 29:19They also showed that a targetable chromatin
- 29:23reader CMI and eight regulates IRF 8
- 29:26by lineage specific enhancers in AML.
- 29:29So at this point we were quite
- 29:31convinced that I of eight might
- 29:33serve as a transcriptional addiction
- 29:35in monocytic leukemias,
- 29:36and we were curious as to how else we
- 29:39can use this in daily clinical practice.
- 29:42What I'm presenting here is work
- 29:44done by Dan Mcquade,
- 29:46who is the 2nd year Yale MD,
- 29:48PhD candidate who really learned
- 29:51all of heme path.
- 29:53It seems within a year and this was
- 29:57just published a couple months ago.
- 30:00We showed that RF-8 specifically stains
- 30:03moneyglass and these extramedullary tumors,
- 30:05so this is a myeloid sarcoma in a lymph node.
- 30:08Follicles are still intact,
- 30:09but you just can see the blast.
- 30:12In the uh, Paracortical area,
- 30:14some of the blocks are more towards medullary
- 30:17area and they were actually MPO positive.
- 30:20Seen here,
- 30:21but the areas of the boss without MPO
- 30:25positivity were expressing IRF 8,
- 30:27so this is in tumor.
- 30:29This is a little dimmer in the
- 30:31mantle zone and when we plotted the
- 30:35relative expression of CD 34 MPO
- 30:37and RF-8 for all of these cases,
- 30:40you see an almost inverse relationship.
- 30:45Another recent I think that we did was
- 30:50looking at BPDCN, so BPDCN is another
- 30:54extramedullary hematopoietic tumor.
- 30:56It's often difficult to diagnose
- 30:58as it presents first in the
- 31:01skin or soft tissue and the most
- 31:04helpful marker to date is CD 123,
- 31:07which is also a druggable target.
- 31:09This can be very dimly staining.
- 31:11This is not just in our lab,
- 31:12it's universally known that city 123
- 31:16can be somewhat dim in this tumor.
- 31:18That really, really relies on the marker,
- 31:21so we look to see if IRF 8 can be
- 31:23helpful in these instances and it was.
- 31:25This was done in collaboration with Doctor
- 31:28Gallery Pansy from Dermatopathology here.
- 31:34Doctor Jacqueline Pinkus,
- 31:35who was the health director at
- 31:38Brigham when I was training.
- 31:40She is just a guru in immunohistochemistry.
- 31:44I think she actually discovered
- 31:45how disdain for Kappa,
- 31:47Lambda and tissue back in the
- 31:49day and when she saw our paper,
- 31:51she immediately started doing some
- 31:53double stains and this is leukemia cutis.
- 31:56Double stain with RV in
- 31:58brown and lysozyme in red.
- 31:59So as you might remember,
- 32:00lysozyme stains all of the different
- 32:04maturation stages of monos and histiocytes.
- 32:07And you can see it staining
- 32:09the history of site.
- 32:10Like Sweets,
- 32:11but there are eight is not staining
- 32:14because there are no glass in here.
- 32:16Other potentially helpful double
- 32:18stains are CD34 with RF-8.
- 32:21I think in terms of what if we
- 32:24have a Milo monocytic leukemia and
- 32:27I think Doctor Pinkus is already
- 32:30doing the double stain with City
- 32:32123 to kind of take out the
- 32:34dendritic cells in a bone marrow.
- 32:38We also looked at the TCG,
- 32:40a pattern of expression for IRF 8.
- 32:43The red block boxes are tumor.
- 32:45The Gray is normal, and you can see
- 32:48for AML and DLBCL you have this very
- 32:51dramatic difference in expression,
- 32:53whereas in the other cancers,
- 32:55not as much in the ones
- 32:57here like this is, I think,
- 32:59stomach and testicular germ cell tumor.
- 33:01We're kind of curious as to whether
- 33:03it really did show that difference.
- 33:05We had a tissue microarray composed of.
- 33:08All these different types of carcinomas
- 33:10and it really did not stain for any of
- 33:13them except for one lymphoma that snuck in.
- 33:15This was actually diagnosed by Doctor
- 33:17Jose Costa really back in the day
- 33:20where it was called malignant lymphoma.
- 33:22So of course that prompted me to do a CD
- 33:2520 just to make sure it was in fact a DLBCL.
- 33:28So for the rest of the tumors you can
- 33:29see here that there is some staining,
- 33:31but that is not in tumor,
- 33:33so that may explain the TCGA data.
- 33:38We also looked at the other
- 33:40differential diagnosis in soft tissue,
- 33:41which is actual sarcomas as
- 33:44opposed to myeloid sarcomas,
- 33:46so this is done in collaboration
- 33:48with Doctor William Wong,
- 33:49who had been a mentor of Doctor Gary
- 33:52Pansies when she was in training,
- 33:54and you can see that for the sarcomas,
- 33:56including the ones that are
- 33:57small round blue cell tumors.
- 33:59They're negative for higher of eight.
- 34:02So so far we've been talking about all
- 34:04the myeloid and monocytic differentials.
- 34:07What about the lymphoid?
- 34:08Because we know it also stains for B cells.
- 34:12Well, here is another common
- 34:14problem in hematopathology classic
- 34:15country and lymphoma versus
- 34:17anaplastic large cell lymphoma,
- 34:19and these have overlapping
- 34:21morphologic features.
- 34:23They also have overlapping
- 34:25immunophenotypic features,
- 34:26and we sometimes have to rely only on PAX 5,
- 34:30which is not always.
- 34:32Positive and classic caution lymphomas
- 34:34and can also be amplified in some alcl's.
- 34:36So we took 74 cases of Hodgkin and 15 cases
- 34:39of ALK negative LCL to see how they would do.
- 34:42Obviously all positive ACL we
- 34:44would not have a problem with.
- 34:47And you can see here that even in the
- 34:49PAX five negative Hodgkin lymphomas,
- 34:51you can have dim staining.
- 34:52For IRF 8,
- 34:53we shouldn't be using this in isolation,
- 34:55of course,
- 34:56but in the context of the
- 34:58morphology and other markers,
- 35:00I think it can be helpful since
- 35:02it is dead negative in all ACL.
- 35:05And this is the kind of
- 35:08composite of cases are pacified,
- 35:10negative I of eight positive,
- 35:12some of course are double negative,
- 35:14and here we really have to look
- 35:16at all the different stains and
- 35:18other features that we have.
- 35:20This was also a first author paper
- 35:23for Dan who just got this accepted
- 35:25for publication couple weeks ago.
- 35:27So in conclusion,
- 35:29IFA can be used to detect extramedullary
- 35:31hematopoietic tumors as well.
- 35:33That can be a diagnostic challenge
- 35:36such as leukemia cutis myeloid sarcomas
- 35:39BPDCN its expression is essentially
- 35:41absent in all other solid tumor
- 35:43malignancies that can present as a
- 35:45differential diagnosis here and as
- 35:47a transcription factor that's also
- 35:49important in B cell lineage commitment.
- 35:51It can show some promise in the
- 35:54detection between Hodgkin versus ALCL,
- 35:56which is a real clinical dilemma.
- 36:01Our findings were just recently
- 36:03replicated by a couple labs in
- 36:05Switzerland and Italy, showing that yes,
- 36:08it is a reliable monoblast marker,
- 36:10but they also show it staining BPDCN well.
- 36:16And thanks to Anoj Verma, our resident here,
- 36:19this has been reviewed in the context
- 36:22of other emerging immunohistochemical
- 36:25biomarkers for myeloid neoplasms.
- 36:30So what about our case?
- 36:31Or a patient with CML?
- 36:33How do I diagnose this as our
- 36:35final diagnosis after showing this
- 36:37case around to multiple other heme
- 36:39path faculty members was myeloid
- 36:41neoplasm with increased blasts most
- 36:43compatible with progression to AML?
- 36:45And our patient was started on treatment
- 36:48with PIXIUS which is a liposomal 7 + 3.
- 36:52It's Donna Robinson was cytarabine
- 36:54with a consideration toward
- 36:56associated Gene and venetoclax.
- 36:58She would have gotten that instead if.
- 37:01The age was older and she
- 37:02didn't have the symptoms.
- 37:05So here is our case.
- 37:07When I went back to stain it with our eight,
- 37:10you can see that in fact the
- 37:11areas where we suspected increased
- 37:13loss there was increase in RF-8
- 37:15and in the areas of maturing
- 37:18Trilineage Marquis there was not,
- 37:20so this certainly made me feel better
- 37:22about calling her as evolution to AML.
- 37:26Another recent study also showed I of a
- 37:29as an AML specific susceptibility gene.
- 37:32So here using publicly available
- 37:35databases they show that these red dots
- 37:39representing genes that were essential
- 37:41to AML but not in non AML cell lines.
- 37:45And here what I really want to
- 37:48highlight is that IFA expression is
- 37:51also correlated with poor prognosis and
- 37:53I think for those of us who remember.
- 37:56The FAB classification and little
- 37:58period of time after that,
- 38:00many times when we first diagnosed AML,
- 38:03clinicians will come down and say,
- 38:05but is it monocytic?
- 38:06Does it look monocytic to you?
- 38:08Because then I will consider
- 38:10this as a worse prognosis.
- 38:12And here we have if we consider
- 38:14RFA to be a marker of mono bus,
- 38:16some substantiation of that suspicion.
- 38:19We're no longer asked that because
- 38:21there's more of a focus right
- 38:23now on exactly what mutations
- 38:25and genetic translocations?
- 38:26They have,
- 38:27but I think that that older clinician
- 38:30perspective of when we say monocytic,
- 38:32it's usually worse for them.
- 38:34Still holds true in some way.
- 38:38This also made me think of a prior
- 38:41observation made a couple years ago,
- 38:43which I think is experientially
- 38:45many hematopathologist feel that we
- 38:47have seen certain very prominent
- 38:50instances of which is monoblast elk
- 38:52growth after venetoclax treatment.
- 38:55So in this study they also segregated
- 38:57their patients based on fab
- 38:59classification and found that only the
- 39:02monocytic classifier was predictive
- 39:04of refractory response to Aza Ven.
- 39:06So here.
- 39:07This is a non monocytic leukemia
- 39:10and this is a monocytic leukemia.
- 39:13They talk primary human AML cells from
- 39:16both what they term primitive and
- 39:19AMOLED and in vitro demonstrated lower
- 39:22kill with both phonetic clocks and a
- 39:26combo phonetic clocks with acidity.
- 39:29They also showed this in a violin
- 39:32plot how monocytic disease arising
- 39:34after treatment can be derived
- 39:37from preexisting subclones.
- 39:38In this other patient at relapse.
- 39:43So our future directions include
- 39:45something Poe hands working on with
- 39:48flow cytometric detection of R8
- 39:50and refining that monoblast gate.
- 39:53Doing that in collaboration with OHSU,
- 39:56a multi institutional validation
- 39:57using AI tools that suit your parent.
- 40:00Cherry is leading and I got a lot of
- 40:03helpful consultation from Doctor David Rim
- 40:06and this is being done in collaboration
- 40:09with MGH BWH Upenn, New Mexico.
- 40:13OHSU Cornell and Stanford and there
- 40:15is a focus in this group on wanting
- 40:18to further look at CML and this
- 40:21whole subclassification of CML which
- 40:24is a bit controversial in The Who.
- 40:27Whether this marker can help
- 40:29us hone it down a little more.
- 40:31And I do hope to identify IRF 8
- 40:34target genes in actual am OL samples
- 40:37and in primary human monoblast.
- 40:39And since I have a regulates BCL,
- 40:41two family members maybe 1 pathway.
- 40:44Of venetoclax resistance in AML.
- 40:48So thank you very much for
- 40:50your attention and your time.
- 40:53Thank you so much for the many people
- 40:55who contributed and really helped
- 40:58move this forward to our external
- 41:01collaborators for the project that
- 41:03our that is ongoing and to our
- 41:06colleagues in derm path soft tissue
- 41:09path pathology tissue services this
- 41:11could not have been done without Amos
- 41:15and Laurie and are flow hematology.
- 41:18And biostats colleagues.
- 41:20Thank you so much.
- 41:30We are open for questions that
- 41:33was excellent by the way.
- 41:36From a non humanoid pathologist,
- 41:39I thought the story is riveting.
- 41:43Thank you.
- 41:45May I ask
- 41:46a question Mina that was fantastic,
- 41:48lot of work and you know you have carried
- 41:51through something that you found and
- 41:53it is now evolving into something far
- 41:55bigger than you may have imagined.
- 41:57Initially from the stain that you
- 42:01showed you in the recent case of the
- 42:05case that you're diagnosed with acute
- 42:08or progression to acute leukemia.
- 42:12It looks like the staining.
- 42:16Is was probably more than 20% and
- 42:18there is a graduation of stating
- 42:20there are many nuclei which are
- 42:22dimmer in their expression of ifit so.
- 42:27Do you think as the last mature,
- 42:30the expression level slowly decreases?
- 42:32Is not an all in non phenomena and
- 42:35second question is this is a surface
- 42:37you know marker in some ways right so?
- 42:42It's a it's a nuclear marker
- 42:43nuclear marker, so is it? So can
- 42:46it be targeted for therapies
- 42:47in some ways or no?
- 42:51OK, so great questions Dan Pat.
- 42:54I think for the first one
- 42:56you know in terms of the.
- 42:58Some lighter or some darker that is
- 43:02very important and I think that.
- 43:05In the validation, what was helpful
- 43:07was its correlation with last count.
- 43:09So we were counting all the ones
- 43:12that had any staining and you know
- 43:15obviously this is going to be.
- 43:20You know, if you.
- 43:21Had a stronger dilution you might
- 43:23be seeing more of the background
- 43:25if you had a lesser dilution.
- 43:27So what we titrated to was that point
- 43:31at which we were seeing a percentage
- 43:35blast that were reflective of the
- 43:38actual morphologic blast count.
- 43:40So it is interesting in that a
- 43:44lot of hematopathologist ask,
- 43:46well, does it stain Pro monocytes,
- 43:48and it's such a good question.
- 43:51I don't know how to answer it because.
- 43:53Homicide is a cytologic definition
- 43:55when we see it in the aspirate
- 43:58and there is no great marker that
- 44:01just gets at the Pomona sites.
- 44:03So I wish that I could just stay
- 44:05in and aspirate that had a ton of
- 44:07promyelocytes and see if it did pick them up.
- 44:10The only consoling part of this
- 44:13whole dimmer staining is that what
- 44:16we got it to is in correlation
- 44:18with that gold standard,
- 44:20so we are counting them and I do think that.
- 44:23At some point it is not
- 44:26seeable by light microscopy,
- 44:29but is probably still baseline there by RNA,
- 44:34right?
- 44:34Like if you look at the really
- 44:36mature macrophage you can see in
- 44:38the gene expression profiles that
- 44:39they do have some expression,
- 44:41but we're not seeing it by by our IHC now.
- 44:45That particular case, if you use that state,
- 44:48your counts for the blast would have
- 44:50been much higher than 30%. Then
- 44:52is that right? Or I'm just?
- 44:54Yeah, I think what I showed
- 44:56you though was one foci right?
- 44:58One area where it seemed a little higher,
- 45:00but then just in practice,
- 45:03and I'm not sure this is the
- 45:04right thing to do in practice.
- 45:06By consensus we we typically have to
- 45:09do it through the whole entire core.
- 45:11Yeah, and in terms of whether
- 45:13it's therapeutically targetable,
- 45:14you know it may be,
- 45:15but it is a transcription factor and it is,
- 45:18you know, also important in
- 45:20B cell lineage development,
- 45:21so I think it would be hard
- 45:23to just try to focus on that.
- 45:26You could end up causing a lot
- 45:29of the problems.
- 45:30David, I think you were next with the hand.
- 45:33Yeah, that my question is similar to
- 45:35Dan Potts in a little bit in a little
- 45:37ways I see that you're progressing
- 45:38toward a multi institutional study
- 45:40which will be really interesting to
- 45:42see if this can be carried out by
- 45:44many pathologists at many places,
- 45:46but it worries me that the intensity
- 45:48at which excel becomes a positive
- 45:50cell because someone were pretty
- 45:52light and some of them are pretty
- 45:55dark and that probably represents a
- 45:57difference in RF-8 expression levels.
- 45:59Do you have any way to standardize that?
- 46:00Or how are you going to check that the
- 46:02results here at Yale are the same?
- 46:04As your other institutions that
- 46:05you're collaborating with,
- 46:07yeah, that's that's a great question,
- 46:09and we are. You know, as you know,
- 46:13we're trying to do this with some
- 46:15quantitative imaging analysis to see out
- 46:18what cutoff point does it actually show.
- 46:22Kind of a consensus with regard
- 46:25to not just the diagnosis,
- 46:28but a consensus across all 7 institutions.
- 46:33And you know, I,
- 46:36I think that this is I wish we
- 46:38had done this earlier with CD 34.
- 46:40In fact, because all these labs.
- 46:42Doing CD 34 and using that day-to-day
- 46:44to tell you whether the AML still there
- 46:48but we don't know how compatible we are
- 46:51with each other and whether you know
- 46:53if we did some quantitative imaging
- 46:56whether it would actually bring us
- 46:58to a better consensus and tell us the
- 47:00truth that we're not reporting right now.
- 47:03So I and you know,
- 47:05nobody really wants to do the bus
- 47:07quantification themselves by I,
- 47:09so I think it's it's really moving us
- 47:11forward to a point where we can be.
- 47:13More standardized across the board.
- 47:15Yeah, I'm not worried about
- 47:16standardized accounting as much as I am
- 47:18standardizing the biochemistry part,
- 47:20that is the tighter and the stain.
- 47:22That is, how do you know
- 47:23if it's if it's too light,
- 47:25then whether you count it by
- 47:26eye or by an imaging system,
- 47:29it will just not be counted,
- 47:31and that's what I would guess that there
- 47:33will be some institutions that will be
- 47:35lighter than other institutions overall,
- 47:37and then that could throw off the result.
- 47:40Absolutely yeah.
- 47:42Transit control,
- 47:42like you know with estrogen
- 47:44receptor we have intrinsic the
- 47:46ducts inside the normal breast
- 47:48and we absolutely have
- 47:50those internal controls.
- 47:51That's the good thing with him is
- 47:53we almost always have internal
- 47:54control because there's so many
- 47:56other cells in the background.
- 47:57So we're using B cells as the
- 48:00control and also dendritic cells.
- 48:02So I've actually seen
- 48:05brighams immunostain for it,
- 48:07which you're using on the Ventana I believe.
- 48:11And also cornells and also.
- 48:16OHSU UM and and then of course the
- 48:20two international groups so so far,
- 48:23seven groups in the US large
- 48:26academic centers have brought it on
- 48:29board for optimization and I have
- 48:31not yet seen one that was very,
- 48:34you know, significantly different
- 48:36in terms of staining a tissue.
- 48:38They've also sent me their tissues
- 48:41to stain to compare side by
- 48:43side so that has been helpful.
- 48:45It takes a lot of work to see.
- 48:47Whether that question and
- 48:48that answer holds out,
- 48:49though it's a great question.
- 48:52Amarie
- 48:54thank you, just a superb talk and
- 48:57such an elegant presentation of your
- 49:00thought process and the steps through.
- 49:03And so I congratulate you as a
- 49:07busy surgical pathologist for
- 49:09thinking for the thought process
- 49:11and for getting this work done.
- 49:13So many congratulations.
- 49:14I I'm I think a lot about inflammation
- 49:17these days and I notice that I
- 49:20don't know anything about RF 8
- 49:22except I do notice that it is.
- 49:25Important in battling infection
- 49:26and that if you if you're deficient
- 49:29in IRF 8 you have a severe primary
- 49:32immunodeficiency and that it's
- 49:35also now in GW studies,
- 49:38they find variance of IRA or a
- 49:41significant risk for autoimmune diseases,
- 49:44and I just wondered if you had
- 49:46any any thoughts about that?
- 49:48Does that manifest at all in your
- 49:50world and pathology and and I'm?
- 49:52I'm wondering if we can use
- 49:54this antibody as well.
- 49:55As we think about these autoimmune
- 49:58inflammatory conditions.
- 50:01Yeah, I that is such a fantastic question.
- 50:04I really don't know that much about its
- 50:08role in autoimmunity except to say that you
- 50:12know if you take out the monocytic lineage,
- 50:16there will be kind of hell to pay, you know.
- 50:19So I think that it it absolutely plays
- 50:21an important role when it's functional.
- 50:24When it's doing its normal job in
- 50:27terms of infection and inflammation.
- 50:29So we cannot.
- 50:31Completely take it out of function,
- 50:34but only in when it is upregulated in tumor.
- 50:39And I think that's it.
- 50:41Also is confusing to me and I think something
- 50:44that I really want to work on is why in in
- 50:47its normal function it is proapoptotic.
- 50:49But then in tumor,
- 50:51obviously they are continuing to
- 50:53proliferate and live on, so it must be.
- 50:57Maybe it's binding differently in some way.
- 51:00When it's tumor,
- 51:02you know there there have been
- 51:05studies showing its deficiency and
- 51:07DLBCL that I have to read more about,
- 51:09but you know it has manifested
- 51:12different kinds of phenotype,
- 51:14whether it's tumor versus normal.
- 51:16So I hope that others will be
- 51:18interested in working on this because
- 51:21I cannot do all of the different
- 51:23types of studies and IRA.
- 51:25I think it's just really cool and.
- 51:27Complicated.
- 51:30Won't you?
- 51:32I've got two questions. One is,
- 51:35do you think that bone marrow aspirates
- 51:38are going to fall out of favor?
- 51:45So, so I don't think so.
- 51:48I you know it's hard for them to do
- 51:51a good aspirate, but a good aspirate
- 51:53is just worth its weight in gold.
- 51:55It is so important to get that
- 51:58fluid sample that we can use for
- 52:01cytogenetics and molecular and flow.
- 52:03I don't think it would ever
- 52:04really fall out of favor.
- 52:06I do think that.
- 52:07We need a better way to obtain it
- 52:09because it is not just like at Yale or
- 52:12you know this clinic or that clinic.
- 52:15When I've talked to other institutions,
- 52:18this is a major problem in the US and
- 52:21folks coming from outside of the US
- 52:25hematologist trained at other countries
- 52:27usually have been really trained at
- 52:30looking at their own aspirates and
- 52:32seeing whether they're good or not.
- 52:34They're much better at
- 52:36getting an aspirate right,
- 52:37so I think it's partly that
- 52:39the training and the US.
- 52:40Focus more on treatment versus
- 52:44the diagnostic procedure,
- 52:46so there might be a swing in
- 52:48the other direction as we see
- 52:50how important the aspirate is.
- 52:53The other question I have is
- 52:56this drug that works again.
- 52:59That targets BCL.
- 53:01Two BCL two is expressed on
- 53:04other lymphocytes as well.
- 53:06So are there what happens to
- 53:10those lymphocytes in patients
- 53:12that are receiving this drug?
- 53:16Yeah, you know no drug is
- 53:18without its side effects,
- 53:20but I I have to say that venetoclax
- 53:22has reportedly done pretty well in
- 53:25these patients because so much of
- 53:28their lymphocytes are actually really
- 53:31abnormal or myeloid cells are so
- 53:34abundant that they have a depression
- 53:37in normal lymphocytes already.
- 53:39So venetoclax has been shown to
- 53:43really induce that initial remission.
- 53:46The duration of that remission is not long,
- 53:49so that is a problem.
- 53:50And these breakthroughs and relapses
- 53:54happen pretty quickly thereafter.
- 53:56So so that is a real issue.
- 53:58But, you know, I think also we,
- 54:02we think of OK, this stain is high,
- 54:05and so this this drug must work, you know.
- 54:08Well in the one.
- 54:09Sustained highly,
- 54:10but it actually has not worked out that way.
- 54:13So in terms of CLL it's worked really well,
- 54:18but you know,
- 54:19full of your lymphoma is BCL 2 positive
- 54:21and yet it's not as it's not doing
- 54:24as well in follicular so it's not
- 54:26like a one to one correlation either.
- 54:31Thank you Mina. The venetoclax
- 54:34is very interesting.
- 54:35Manju, because this is a drug where you
- 54:38actually have to titrate up in CLL,
- 54:39you give it all at once and patients
- 54:42will get extreme tumor lysis syndrome.
- 54:44The problem with these drugs as well,
- 54:46based on their structure
- 54:49is they're extravascular.
- 54:51Extrusion is very limited,
- 54:52so there are many tumors where within the
- 54:55circulating system it's very effective,
- 54:57but on tissues it's just a drug property.
- 54:59That drug is very,
- 55:01very tightly albumin bound and
- 55:02very poorly able to actually
- 55:04egress from the bloodstream.
- 55:07Thank you, thank
- 55:08you so much. That's a great point.
- 55:11Hi Mina, I have a question for you.
- 55:15So this is more of an
- 55:18immunohistochemistry question.
- 55:19Are you really looking into multiplexing?
- 55:22And since this monoblast count
- 55:24is so critical in the 20% count,
- 55:27so I was wondering,
- 55:28are you relying only on IR F8 and would
- 55:32you also consider multiplexing with CD 34?
- 55:35But then we no CD 34 doesn't pick
- 55:38up all monoblast monocytic leukemia,
- 55:40so is there any other marker for example,
- 55:44which is cytoplasmic localization?
- 55:47Like CD 117 would be more helpful
- 55:49for you versus CD 34, which also I
- 55:52believe goes to the nucleus, right?
- 55:54So having a Multiplex stay in that is.
- 55:57You know,
- 55:58picks up two different localizations
- 55:59would be more helpful than having.
- 56:01Absolutely yeah,
- 56:03I'm I'm so glad you brought it up
- 56:05and I really hope to get your buy in
- 56:07and doing the multiplexing. In fact,
- 56:10I think CD34 with RA would be perfect.
- 56:13In fact, CD 34 is going to be membranous,
- 56:16and this is nuclear.
- 56:17Then we would capture all of them, you know.
- 56:21And at MGB they're already doing 123 with RH,
- 56:26just to show its double.
- 56:28Expression in the dendritic cells,
- 56:32so so that's another possibility.
- 56:34But I I do think that in cases where
- 56:37we really are relying on this kind of
- 56:41getting to 20% the the multiplexing
- 56:43with 34 would be super super helpful.
- 56:46So I'd be happy to work on it
- 56:48with your staff.
- 56:53Thank you so much for all of your time.
- 56:57Please feel free to ask me any other
- 56:59questions as you might see me in
- 57:00the hallway. Thank you, thank you,
- 57:04thank you all for coming.
- 57:07Great talk.