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INFORMATION FOR

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.