Pathology Grand Rounds: April 6, 2023 - Abner Louissaint, MD, PhD

April 06, 2023

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Translating Molecular Characterization and Preclinical iInvestigation of B-cell Lmphomas into Impactful Therapeutic Opportunities - by Abner Louissaint, MD, PhD

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00:00All right. It is my great pleasure
00:03to introduce Doctor Abner Lucent
00:06for our grand rounds today.
00:08Doctor Lucent is a hematopathologist
00:10at MGH and associate professor
00:12at Harvard Medical School.
00:14Upon graduating from college at Wash U,
00:16he went to Cornell for his MDPHD,
00:18followed by a PCP residency and
00:21then Heme Path Fellowship at MGH.
00:24He has since been there,
00:24rising to the rank of associate professor.
00:26He is currently director
00:28of the Hematology Lab.
00:30As well as the nascent
00:33lymphoma tissue repository, Dr.
00:35Luson has characterized novel
00:37subtypes of follicular lymphoma
00:39such as pediatric type follicular
00:41lymphoma and has defined the genetic
00:44underpinnings of these tumors.
00:45So specifically,
00:46his initial study in blood
00:48demonstrated the genomic differences
00:50between pediatric type and the
00:53traditional follicular lymphoma,
00:54representing a major advancement
00:56in the field.
00:57On the basis of this work
00:59and also follow up studies.
01:00PD type molecular lymphoma is
01:02now a distinct entity in The Who.
01:04More recently,
01:05he has characterized the genetic
01:07landscape of additional lymphoma
01:09subtypes including primary
01:10duodenal follicular lymphoma,
01:12primary cutaneous follicle
01:13center cell lymphoma,
01:15primary cutaneous gamma delta T
01:16cell lymphoma, as well as DLBCL.
01:19Leg type Doctor Lusan's lab has
01:21established the first ever PDX
01:23model of follicular lymphoma.
01:25He is currently an author for
01:27five chapters of the upcoming
01:285th edition of The Who and lead
01:31author for four of these chapters.
01:33He's highly involved in ash,
01:35working on the Publications committee as
01:37well as the Abstract Review Committee.
01:39He has won the Benjamin Councilman Award,
01:41outstanding paper and pathology
01:43through use CAP,
01:44as well as the Berard Dorfman Founders
01:46Award for Young investigators
01:48through Society for HEMATOPATHOLOGY.
01:51I knew of Abner,
01:52but then got to know him personally
01:54through a rather epic study that is ongoing,
01:58and for that I got tasked with
02:00evaluating 789 potchkin lymphomas
02:05and I thought.
02:07Nobody would be willing to work
02:09on this with me,
02:09particularly someone with a lab himself.
02:13But Abner has proved me wrong and has
02:17helped greatly in that translational study.
02:19What I didn't know about him until
02:22very recently is that he started
02:24his academic faculty position
02:26focusing on clinical hematopathology.
02:28And was not in fact in the lab when
02:30he asked those fundamental questions
02:33about pediatric type follicular
02:35lymphoma and that resulted in his Kay Ward.
02:37He later carried out these
02:39experiments in David Weinstock's lab.
02:41And I think it is this unique pathway
02:43that he has carved that demonstrates
02:45his scientific queries are truly
02:47grounded in his own personal clinical
02:49expertise and that is something
02:51that I find truly inspiring.
02:52So thank you so much for coming
02:54to speak at Yale Grand Rounds.
02:57Thank you so
02:57much for that really kind introduction
03:00and it's it's truly an honor and
03:02pleasure to be here in the department
03:05and thank Doctor Chu and Doctor Lu
03:07for hosting me in the department.
03:09I'm really happy to talk a little bit
03:11about some of the work that we've done
03:16just I have no disclosures and.
03:19I will talk about different,
03:22different efforts,
03:22but they all center around a
03:25central goal which is if you think
03:27about lymphoma or what we do as
03:29pathologists and classifying the
03:31over hundred types of lymphoma.
03:51And all the work that I'll be
03:53presenting has attempted to do is to
03:56identify biomarkers that can either
03:58predict or give us a sense of help
04:00us understand how different lesions,
04:03why different lesions respond differently
04:05to therapeutic interventions and the
04:07heterogeneity responses that we see
04:09even within a single disease entity.
04:12And then occasionally when you
04:13do the investigation,
04:14you end up finding that what
04:16was thought to be a part of an
04:19entity is actually its own entity.
04:21And so with that I'll start,
04:23I'll start each of these sections with
04:25sort of a clinical case that sort of
04:28represents the impact or of the work.
04:30So the first is a 25 year old man with
04:34isolated cervical lymph adenopathy
04:36limited stage with clonal CD10B cell
04:38population by flow cytometry and
04:40you can sort of see a confluence of
04:43expanded follicles and the sort of
04:45largest medium to largest sort of cells.
04:48And there's an architectural pattern to it,
04:51focular pattern.
04:52The neoplastic cells are CD10 positive,
04:55their BCL two mostly negative and have
04:57a really high proliferation fraction.
05:03And so traditionally when this was
05:07originally diagnosed, this case,
05:08it was diagnosed as a focal lymphoma and
05:10I'll talk a little bit about that for those.
05:13Not in familiar with lymphoma,
05:14but it was diagnosed as a high grade Grade
05:183 focal lymphoma and it was limited stage.
05:21And so the question there is do you
05:24treat with chemotherapy at the time
05:26or do you observe radiation therapy?
05:28And so we'll get back to the case,
05:31but just as a background focal lymphoma
05:33is a neoplasm of germinal center B cells
05:35comprised of centrocites and centroblasts,
05:37normal cell types within follicles,
05:40lymph node follicles.
05:41This disease demonstrates a
05:43policular growth pattern.
05:44The mean age is 6 decade and often
05:47presents with advanced stage disease,
05:50usually involving lymph nodes
05:52can occasionally involve marrow
05:54and extranodal sites.
05:56These follicular lymphomas can have
05:58different contributions of centrocytes,
06:00which are smaller cleave cells
06:03and larger centroblasts and today.
06:10Grade one to two would be sort of a lower
06:13grade and tends to have these smaller cells
06:16with sort of irregularly shaped nuclei.
06:19Whereas the central blasts are larger,
06:22more round with usually nuclear that
06:25are opposed to the nuclear membrane
06:27and 3A and 3D split between 3D
06:30being sheets of these large cells,
06:323A being more than 15 per high power field.
06:35And generally the thought is
06:37that the grade threes are.
06:39May have a behave worse and may it may
06:45require more aggressive chemotherapy
06:47and the current upcoming WHO grading
06:52has been removed and and and the ICC
06:55classification it's still there but
06:57for the purposes of this just want
06:59to present the difference so these
07:03focalformers have a fundamental genetic
07:05alteration which is BC L2 translocations.
07:09Which the 1418 which juxtaposes BC
07:12L2 upon regulatory enhancer elements
07:14of the heavy chain which causes
07:17up regulation of BC L2 expression
07:20which imparts survival advantage
07:22and it clinically we can see this
07:31expression representing a germinal
07:32center cell and you can see that in.
07:38Folk lympharma, traditional classic,
07:40folk lympharma, you have BCL two
07:42expression and traditionally the most
07:46folk lympharmas have a relatively low
07:49proliferation fraction and usually
07:51a reactive general center will have
07:53a very high proliferation fraction.
07:55And we can do additional studies to look at
07:58clonality like PCR for IGHD arrangements,
08:01fish for for to assess the the BCL
08:052 rearrangement and flow cytometry.
08:08Now early on we had identified there
08:11were some early series looking at folk
08:13and plumber and children and there
08:15there was some common trends noticed.
08:17So many of these young patients
08:21were were boys, young young boys.
08:24And there was they presented with
08:25limited stage disease often in
08:27the head and neck region,
08:28often had what was thought to be high
08:31histologic grade like more like a Grade 3
08:33or thought to be and they often lacked BC L2.
08:37Next question,
08:38but interestingly in all these
08:39series there was durable remission.
08:41Often these patients they'd get
08:43chemotherapy but sometimes they
08:45did not and and response was good.
08:49And so back in 2008 this was considered
08:51a provisional entity pediatric
08:53cochlemphoma with where you have these
08:55folkformers and kids that did well,
08:57they tended to have what they called
08:59grade 3 morphology expansive follicles,
09:02but it was clear that these
09:04pediatric cochlemphomas.
09:05Had many features indistinguishable
09:06from those seen in adults.
09:08And so at the time when I answered the field,
09:10there were some questions that came to mind.
09:12One is that as we got more
09:15and more experience,
09:15we realized that many of these patients
09:17had no progression or occurrence
09:18even with just excision of the node.
09:20And I began to see a lot of these cases
09:23presenting in patients with in their
09:2520s and 30s with the same features.
09:28So now the question is,
09:28are are these Grade 3 folliculars
09:30or the pediatric folliculars?
09:32And so we asked the question how
09:33can we actually distinguish these
09:34because it's going to actually
09:36make a big difference in care,
09:38how can we objectively define these.
09:39So we started by looking at 27,
09:42you know all the focal point of
09:44patients at MGH that were less than 40
09:46years of age and we found that they
09:48should have broke into two groups,
09:49one that were limited stage and
09:52one with advanced stage disease and
09:54the ones that were limited stage
09:56we did see a predominance in in in
09:59a male predominance.
10:01And we looked at a whole slew
10:03of pathological features to
10:05try to distinguish those.
10:06We did find that the the limited
10:08stage ones had large follicles
10:10and had a star sky pattern,
10:12but many of the other parameters
10:14didn't pan out to make a difference.
10:16But one thing that we noticed really made
10:18a difference was the BCL 2G arrangements.
10:20So all of the limited stage ones lacked BCL
10:23two arrangements and BCL 6 arrangements,
10:25and had a very high proliferation
10:27fraction greater than 30%.
10:29And they had the combination of them
10:31whereas the the Advanced Age ones,
10:34the majority did not have none of them
10:36had both and both of those features.
10:41So we thought well maybe this is something
10:43maybe maybe these two features the the,
10:45the BCL 2 gene arrangements and the
10:48proliferation fraction together could
10:49could pick these good behaving cases out.
10:52So then we looked at a second cohort of
10:54adult patients less than 40 years of age.
10:57Right. And we've broken them up
10:59into four categories, you know,
11:01translocation, no translocation,
11:02no translocation and high proliferation,
11:04low proliferation index and the
11:06ones that had no translocation
11:08and high proliferation fraction,
11:10all of them ended up being stage one disease.
11:14Many of them did get chemotherapy
11:16and in terms of progression relapse,
11:18none of them had progression or relapse.
11:20And actually I remember reading the notes
11:22and it would be like these surprising notes,
11:23Oh my gosh,
11:24this is patients doing really well and.
11:27And but this trend was really important
11:29to us and this is just a looking at
11:32Kaplan Meyer sort of curve showing
11:34the differences between the the cases
11:36that have BC L2 arrangements and High
11:38proliferation index and the ones that didn't.
11:40At the same time Elaine Jaffe's
11:43group described that these these
11:45pediatric follicular lymphoma cases.
11:49Had a very different morphology.
11:50So they're the morphology was not
11:52that of typical Centra blast where
11:54you can see these larger cells with
11:56these nucleoli sort of centers sort
11:59of touching nuclear membranes.
12:01They were more of a medium sized
12:03blastoid type phenotype and for that
12:05reason suggested that we shouldn't
12:07grade these these these cases.
12:08And when you look when we looked at
12:10all of our case at MGH we we found
12:12that there were two sort of peaks,
12:14so the pediatric type focal from a peak.
12:16Peaked in adolescence,
12:17but you can see that it tailed
12:20into adulthood versus.
12:23They took the classic focalforma which
12:25as we know peaks in the in the 6th,
12:277th decade,
12:28but it's in this intermediate range
12:31where it becomes important to be
12:33able to distinguish the difference.
12:37So from this we proposed that there was a
12:39highly indolent subset of focalforma which.
12:42Occurred in patients which were not likely
12:44to progress and did not require chemotherapy.
12:47Characterized by the lack of the B,
12:49CL2B, C,
12:49L6 arrangements and high
12:51proliferation fraction,
12:51and we hypothesize that they were
12:55biologically distinct and common in
12:57adolescents and young adults,
12:58but can occur in older patients.
13:01At the time,
13:02we were really excited about this,
13:04but we realized we saw a phenomenon that.
13:08You know,
13:08we hadn't really,
13:08we they were histologically similar
13:10to high grateful lymphoma in some ways
13:12and there was no direct evidence that
13:13the ones that occurred in adults were
13:15equivalent to the ones that occurred in kids.
13:17And practically speaking,
13:18we noticed,
13:19I noticed that a lot of patients
13:21who presented young patients,
13:23it depended their,
13:23their therapy depended upon who they saw.
13:25So if they saw a pediatric pediatric
13:27oncologist they would be quickly,
13:29they would be observed and
13:31they would do fine.
13:32And if they saw an adult
13:34oncologist they'd basically be
13:35given our chop and they would do fine.
13:37So we I thought it was important to go
13:39further to define these objectively so that
13:42basically to avoid unnecessary therapy.
13:45And so the question hypothesis is
13:46that pediatric type filial fund
13:48is biologically distinct and we
13:50wanted to look at the mutational
13:52profile differences between the two.
13:53Now to do this,
13:54we had to leverage colleagues from
13:56different institutions across the
13:58country to get these uncommon cases.
14:00So we put together 44 cases
14:03of limited stage disease,
14:04so we were not including
14:06advanced stage disease,
14:07no DLBCL and basically split them up
14:10into pediatric type focal components
14:11defined by the BC L2 rearrangement and
14:14high proliferation fraction versus the
14:16ones that either had BC L2 arrangement
14:18or locally low proliferation fraction.
14:22And we found when we did this again that
14:24the the ones that we call pediatric
14:26type had a had a male predominance were
14:29mostly involving the head and neck.
14:31And when we looked at the ones above
14:33older than 18 or younger than 18,
14:35which is the classic definition of pediatric
14:38and how sometimes these were defined,
14:40they had a similar breakdown male
14:42predominance and head and neck predominance.
14:45And in terms of therapy,
14:47a lot of the limited stage
14:50ones did get chemotherapy,
14:52the classic folk problems did
14:54get chemotherapy and radiation.
14:56A lot of the ones that were
14:58called PTFL did get excision,
15:01but the sort of rates were very different.
15:03So the the patients,
15:05the PTFL patients did really well with
15:08with remission no evidence of disease.
15:12Whereas the the the ones that were
15:14more classic had a BC L2 arrangement
15:17or high a low proliferation fraction
15:19were much more likely to recur and
15:22transformation events occurred
15:23solely in that group.
15:24This is a Kappa Meyer and when you
15:27looked at the breakdown between
15:29pediatric type in older patients
15:31and and less than 18 or older than
15:3318 the the the the the same trends
15:37occurred in terms of doing well.
15:40So we then looked at the the the
15:43molecular dynamics of these lesions
15:45and we found that the pediatric
15:48type colliculum informas had a very
15:51low genomic complexity with you
15:53know much fewer genome copy number
15:56alterations and genomic events
15:58than the typical for lymphomas.
16:00And there was a high higher predominance
16:03of loss of heterozygosity 1P36 and
16:05deletions at that site of TNFRSF 14.
16:09But the most striking thing was
16:11when we looked at the pediatric
16:14type whether lower than 18 or older
16:16than 18 that the classic.
16:18So we we did a targeted panel that
16:20included pretty much every gene
16:22that had been mutated reported to be
16:24mutated in focal form at the time
16:27and which was much more than here almost 100
16:34I think 100 genes.
16:35These these on the left are sort
16:37of the ones that are classically
16:39mutated in full lymphoma.
16:41And you can see that the even limited
16:44stage ones that had BC L2 arrangement
16:46or low proliferation fraction had a
16:48high frequency of these mutations.
16:50But they were essentially
16:52not present in the PTF,
16:53the pediatric type full lymphomas other than
16:58TNFTNFTNFRSF 14.
17:01We also did helexome on a on a on on
17:03six of the cases and we didn't find
17:05any recurrent mutations at the time.
17:07And this histogram just shows the purple
17:10and the blue represents 2 big subsets
17:12of typical classic focal FOMA and the
17:15most common mutations including ML,
17:18L2, probably PA,
17:19lot of chromatin modifying genes.
17:21And you can see that the the low
17:23frequency in the pediatric type
17:24focalforma other than the Tina,
17:25Tina, RSF 14.
17:26So we thought that they suggested
17:29definitely these were biologically distinct
17:32and we were pretty happy with that.
17:35And but we weren't clear what was driving
17:37the the pediatric type proliferation
17:38but at the same time we submitted
17:40to to blood and we're very excited.
17:42We we received a review saying well you
17:45only looked at six cases of whole EXIM.
17:47We think you should look up more
17:49and that was a little depressing
17:51at the time but it was turned
17:53out to be very fruitful and maybe
17:55appreciate the review process because
17:57we we basically sequenced all,
17:59the whole EXIM and all we found that.
18:01About 60% of these cases had
18:03mutations in the MAP kinase pathway,
18:05particularly MAP 2K1 at a
18:08a targeted hotspot site,
18:10which is present in this negative
18:14regulatory region of map 2K1 and represents
18:17a mutation that's commonly seen in
18:19other neoplasms including melanomas,
18:21****** hand cell hysterocytosis,
18:23B rap negative hairy cells,
18:26etcetera.
18:26So we were very excited about this and
18:28we thought this clearly demonstrated
18:30that these were biologically distinct.
18:33And at the time three groups were
18:35working on this simultaneously.
18:38And this is sort of a summary of
18:40all the cases in those three groups.
18:43You can see that again the common
18:46mutations typical and full lymphoma
18:48are very lowly are very rare in
18:51pediatric type and the map 2K1 and
18:53the MAP kinase pathway mutations
18:55really seem to be to drive the
18:57pediatric type molecular ones.
18:59The the one that is shared is
19:01the mutation of TNFRSF 14.
19:03So from this we you know we use
19:06the term pediatric type lymphoma
19:08to to include the fact that these
19:11occur in older patients.
19:12And you know from that that sort
19:15of helped define the current our
19:17current understanding of pediatric
19:19type of lymphoma and and in the
19:21DOUBLECHILD 2016 that was the help
19:24define the the characteristics.
19:26Some of the important features are
19:27that grading is not used for these
19:30for the reasons I mentioned there's
19:32never any advantage disease and
19:35there's no component of the LBCL.
19:37So if there is a if you see the
19:39LBCL that excludes this diagnosis.
19:41And the clinical implications that
19:43both children and adults with PTFL
19:45do not likely need chemotherapy when
19:47they're when they're diagnosed that
19:49way and the map kinase mutation may help,
19:52may help with the diagnosis.
19:53So going back to the patient I
19:55described that was originally
19:56diagnosed as grade three O 3,
19:58this was re diagnosed by Elaine Jaffe
20:02at at, at, at as pediatric type
20:05for lymphoma but at the site where
20:08the patient was being treated.
20:09They were still going to give
20:10our chop because they weren't
20:11familiar with this entity.
20:12And so I received a call from the
20:15mom about this and had seen the paper
20:17and I refer them to our ecology team
20:20who who basically were suggested
20:22observation and the patient's doing
20:24well and I still occasionally get calls
20:26from families about this where the
20:29the thought is to treat aggressively
20:32and they're always actually happy
20:34to to to feel like I've made some
20:37impact on the care of these patients.
20:39I just wanted to emphasize that
20:41it's really important to to follow
20:43all the criteria and this this
20:47case sort of demonstrates why.
20:49So this is a 25 year old with
20:52cervical lymphadenopathy,
20:54the nice space dot star sky pattern
20:56and the sort of blastoid type
20:59cells and these are CD10 positive
21:03with B cells with a relatively
21:06high proliferation fraction and.
21:09No BC L2 expression.
21:10So this was brought to me as a as a
21:12consult as whether this could be a
21:14nice classic case of pediatric type
21:16lymphoma and I asked you know did
21:18you do the fish for B CL2B C L6 and
21:21they didn't and it was frustrating
21:23for them but it after came back
21:26with ABC L6 gene rearrangement.
21:27So this is essentially a conventional
21:30BC L2 negative lymphoma.
21:32So it's not a pediatric type
21:34which and these do occur.
21:37And interestingly later there was
21:39you know months later there was a
21:40axillary lymph adenopathy and they
21:42re biopsied it because they were a
21:43little concerned by the diagnosis
21:44that it was not called pediatric type
21:46this time the the Falcons were much smaller.
21:50There was this eosinophil deposits and
21:53deposits that looked a little bit like
21:55Dutcher bodies and some of the cells,
21:57the cells were relatively small.
22:01The follicles were or small smaller
22:03again CD10 positive but this time
22:05the proliferation fraction was low
22:07and there was BC L2 expression.
22:09So clearly a case of classic
22:12classic folk lymphoma.
22:14So it's really important to follow
22:15all those guidelines and make
22:16the diagnosis of pediatric type.
22:18It's not just the BC L2 negativity.
22:22So from that you know we looked
22:25at several subtypes of folk
22:27lymphoma to to try to understand.
22:29Them and sort of the variation in in
22:32their behavior and I'll just mention
22:34while the other one which is the primary
22:37utaneous follicle central lymphoma
22:38which are localized skin lesions.
22:41They by definition don't are not
22:44systemic disease and they often
22:46occur in the head and the trunk.
22:48They have an excellent prognosis.
22:50Again they're negative for BC L2,
22:52they lack BC L2 rearrangements.
22:55So they're clear similarities to
22:57to pediatric type of lymphoma.
23:00So we wanted to investigate to see if
23:03we can understand understand these.
23:05We did, we did a study where we
23:09basically got primary continuous,
23:13continuous focal pharma and and
23:16secondary primarily being ones
23:18that occurred in the skin and
23:20actually and and not systemically.
23:22Initially there were four cases
23:23and we occasionally see this where
23:26it it fulfills the features of of.
23:28A primary utanous follicle center lymphoma,
23:30but then years later, months later,
23:32years later has systemic dissemination.
23:34So we had four of those.
23:36And then we also had secondary utanous
23:39follicle center lymphoma where there
23:41was a previous systemic disease
23:42involving the skin or they currently
23:45presented systemically and in the skin.
23:47And so the hypothesis that these
23:50would be genetically distinct and
23:52there may be initial differences.
23:55So you can see here that the median range,
23:57age range was approximately
23:59similar across these groups
24:03and in terms of therapeutic therapy
24:08was pretty it was pretty similar
24:11as well and the the patients with
24:13primary continuous fossil center
24:15of course did much better and but
24:17they they did they did recur as
24:20these as these cases do recur but
24:22the patients still do very well.
24:24One of the interesting things we noted
24:26was that the recurrences tended to
24:29occur at the similar same location.
24:32So you can see.
24:33You can see that here where the
24:35recurrences occurred pretty much at the
24:38same sites as the primary location,
24:40even the secondary recurrences.
24:42Whereas the the four cases that
24:46actually eventually recurred in a
24:50in a different site or were more.
24:52Groups presented systemically
24:54had quite interesting locations
24:56such as the dura breast and
25:02bone marrow, and so one of
25:05the things we noted was that
25:07these secondary cutaneous folk,
25:09the secondary cutaneous folk lymphomas,
25:12harbored a lot of the mutations
25:14in the chromatin modifying genes.
25:16Whereas again the primary cutaneous had
25:18fewer although they were more they were they,
25:21they occurred more frequently.
25:22I mean they they they had there were
25:25more cases than in primary pediatric
25:27type and that that had these but they
25:30were definitely much fewer than the
25:33secondary cutaneous folk lymphomas,
25:35the few cases that actually presented
25:39like primary cutaneous falcal,
25:41center lymphoma but then.
25:44Showed systemic involvement
25:46interestingly showed
25:49more frequent mutations in these
25:52chromatin modifying genes.
25:55So we proposed an algorithm to try to
26:00predict cases or to to help support
26:04cases that we that would potentially
26:07behave either at the time of diagnosis
26:10or later with systemic disease and.
26:13Basically the way it works is 1
26:16criteria is having both mutations in
26:19KMTTD and Kreb BP or having more than
26:23three chromatin modifier gene mutate,
26:25more than 3 mutations and chromatin modifier
26:28genes or the presence of BCLTG arrangement.
26:31And we found that if you had more than two
26:37of these two or more of these criteria
26:39that there was a very high risk.
26:41That this represented a lesion that
26:44was going to behave more systemically.
26:46And just to demonstrate this,
26:49these are few cases.
26:50This is 1 case of Prime Minister
26:53Falcon Center Oklahoma with that was
26:56had no BC L2 generated in the 74
26:58year old in the scalp and this had
27:01classic there was CD20 positive cells,
27:04somewhat of a high proliferation fraction.
27:07Again the cells were BC L2 negative.
27:09There was a relatively high,
27:11like I mentioned,
27:13proliferation fraction and there
27:15was no mutations in these in the
27:17common chromatin modifier genes.
27:19There was no B CL2B C L6 arrangement
27:22and this patient to the end of follow
27:25up never had any systemic disease,
27:28but there was.
27:29Here's another case of a 47 year
27:31old male who had a forehead lesion.
27:35The I HC showed terminal Center B cells,
27:37but these were BC L2.
27:40Positive there was no BC L2,
27:43B CL6GG arrangements.
27:45It was signed out as possible cutaneous
27:48falcocenter lymphoma primary but these
27:51this turned out to have mutations
27:54prebby P and KTM2D No B CL2B C L6
28:00arrangements and then imaging at
28:02the time showed concurrent nodal
28:04involvement and then there was a 45
28:06year old with a male scalp lesion.
28:09Again,
28:11no B CL2B C L6 arrangement
28:13signed out as possible primary
28:15cutaneous Falco Center lymphoma.
28:17Again this had Kreb BPKTMTD
28:21mutations and ABC L2 arrangement.
28:23The imaging at the time should not
28:25no concurrent nodal involvement,
28:27so it was thought to be a primary
28:29cutaneous Falco center lymphoma.
28:31But thirteen months later there was
28:32involvement of the bone marrow and
28:34axillary lymph node involvement.
28:35So I think,
28:36I mean there's still has to
28:37be validation for this,
28:38but I think these parameters can help
28:42in what can sometimes be a difficult,
28:46in some difficult cases and
28:47trying to predict,
28:48maybe even help support when
28:51these patients should be followed
28:53carefully for systemic disease.
28:55So to summarize some of these
28:59subsets that I've described,
29:00you have classic focal Pharma with B,
29:02CL2B, C L6 arrangements.
29:03And krubby P mutations and KTMTD mutations,
29:06other chromatin modifying G mutations
29:08with a low proliferation fraction.
29:11And then you have which is
29:13classic focal FOMA.
29:14You have pediatric focal FOMA
29:16which lacks the BCL two BCL 6 and
29:18lacks these chromatin modifying G
29:20mutations but has kinase mutations
29:22has a high proliferation fraction.
29:24These are most likely not arising
29:27from the typical.
29:28Recursive cell in the bone marrow
29:30and where which derives the
29:31systemic form of this,
29:33but it's probably represents
29:35a locally stimulated clonal
29:37follicular proliferation and
29:38similarly primary containers.
29:39Falconceno FOMA is is most likely
29:42locally stimulated focal lymphoma,
29:44but a subset of these that appear
29:47to be this way may actually be more
29:50have a more of a systemic behavior
29:52and I think looking at these
29:55mutations these underlying mutations
29:56may help predict those few cases.
29:59So you know,
30:02one runs against the one runs against the
30:05wall when really trying to identify
30:08biomarkers with basically pieces
30:10of tissue and characterizing,
30:12we can look at mutational profiles,
30:14but I began to think about ways that
30:18we can get around that in terms of
30:21looking at snapshots of mutational
30:24landscapes or expression profiles.
30:26Wanted to do more functional,
30:28be able to look at the more functional
30:30behavior of these cells in a context
30:32of microenvironment or be able to
30:34assess responses in therapy directly.
30:36And so began to think about models
30:38to do this and we started to develop
30:41patient derived xenograft models and
30:43we initially developed a repository
30:45where you know the lymphoma cases
30:47go to the frozen lab and we have a
30:50pipeline where tissues that are large
30:53enough that are not small biopsies.
30:55We can set aside some tissue and
30:57viably freeze that tissue and consent
31:00the patients and then in a very non.
31:03So we're not targeting any particular
31:05Histology or anything like that.
31:06We've done this for about 200 cases to
31:10date and it's been a very fruitful effort.
31:13It allows us because we're not
31:15going after specific histhologies
31:16to occasional to capture rare cases
31:19that if you were going to try to
31:21target that would be hard to do.
31:24And diagnostic challenging cases
31:26and the the viably frozen being able
31:29to viably freeze them allows us to
31:31then thaw them out and generate
31:33PDX models or try to culture them.
31:36So it's been great.
31:37So that's sort of our resource for this.
31:40And then the way we generate these
31:43PDX models is we use NSG immunity
31:46deficient mice and we initially we
31:48take those little pieces of tissue
31:50which you can see on the.
31:52On the on the right here that we
31:54viably freeze and we implant them
31:55underneath the capsule,
31:56the the renal capsule.
31:58So we pop out the kidney,
31:59we make a little hole and we stick the
32:01tissue in there and then eventually it
32:04grows into something on the bottom now.
32:06So you can see it just becomes a tumor
32:09and we can then take that and we
32:11can keep propagating that over time.
32:14And these models are very helpful because
32:15you can use them to test different therapies.
32:18And you basically are growing more
32:20tissue that you can then study live
32:25and so we use this recently for in in a
32:29more aggressive disease and again I'll,
32:31I'll I'll start with the case of
32:34a 42 year old man presented to the
32:36Ed with junction night sweats,
32:38anemia and basically a lot of
32:42disease lymphadenopathy.
32:43A biopsy was rendered and basically
32:47we have these sheets of very large
32:50cells with a lot of areas of necrosis
32:54and apoptosis and the cells had were
32:57large and had plasma blastic sort
33:00of phenotype with very prominent
33:02nucleoli and essentially placed nuclei.
33:05The cells were strongly out positive
33:08and lacked CD20 and CD3B cell and
33:10T cell markers and had some CD138.
33:14So essentially this was the diagnostic
33:17of positive large B cell lymphoma
33:20which is driven by overexpression of
33:23anaplastic lymphoma kinase which is
33:26which was reported fairly recently in 97.
33:29These are extremely rare lymphoma cases.
33:32They often do express plasma
33:35cell plasoblastic markers.
33:37They lack typical B cell T cell markers.
33:40The patients tend to be young,
33:41the median age.
33:42In the 40s, one third of them
33:44are in the pediatric age group.
33:46The the patient, the the prognosis
33:48is dismal and many patients die,
33:50especially in advanced stage
33:51with within a few years.
33:53And because of the rarity of the disease
33:55and the aggressiveness of the disease,
33:58it's essentially impossible to
33:59to set up a clinical trial.
34:02The the ALC anaplastic lymphoma kinase
34:05is a receptive tyrosine kinase originally
34:08discovered in anaplastic large cell
34:10lymphoma which is an op positive.
34:12T cell from a 94 and these
34:15are driven by fusions.
34:16The alcos of a LC L's driven by
34:19NPM fusions with with the kinase
34:23part of the ALC protein.
34:25But the alcosa large B cell from us
34:27tend to have the partner tends to
34:30be clathrin so different than the
34:32ALC L's and these lead to downstream
34:34signaling and Jack stat and P cell
34:36gamma and Ras irk Pi through kinase pathways.
34:42But you can see that the prognosis
34:44is pretty dismal for patients that
34:46particularly in advanced stage disease
34:50and there has been as you probably
34:53know ALK inhibitors developed for
34:55these malignancies all kinds of lung,
34:58all kinds of with without fusions and
35:01that have been fairly effective so
35:05soon after the discovery of these fusions.
35:08The the first generation OP inhibitor
35:10was was was generated and and and
35:13shown to be effective in in different
35:17positive neoplasms and then then
35:19there was second and third generation
35:21inhibitors that had higher potency.
35:23So interestingly in in AL positive
35:27anaplastic large cell lymphoma
35:28there has been that you you get a
35:31significant response right and to
35:33this to chrosatini which is the
35:35first generation OP inhibitor but.
35:36Where the out positive large B cell
35:38from is you actually these patients
35:41often go through a several sequential
35:44highly aggressive chemotherapy
35:45regimens and don't do well.
35:48And anecdotally people have tried
35:50the chrysotonyb and basically the
35:52patients have maybe had partial
35:54response and then essentially failure.
35:56And so the few cases that have been
35:59reported in the literature anecdotally.
36:01Have have have shown like a a
36:04very short response,
36:05a transit partial response and then
36:07failure and that's only been a couple.
36:10So that that had been the status of the
36:12field and these patients just don't do well.
36:15So the rationale for PDX modeling
36:17is that you know to to look at the
36:20efficacy of these OP inhibitors and
36:21the the fact is we can't do clinical trials.
36:23So could we use these models to test
36:26these OP inhibitors in this disease.
36:29So again this is just another image
36:32of how we make these.
36:33So you can see the small tumor
36:35and and basically
36:38we pop that back in and then we
36:39generate a tumor on the kidney,
36:41this actually is a tumor and out tumor.
36:45So on the left here you can see the
36:47normal kidney on the right these
36:49go pretty quickly and you can sort
36:50of see the size of the tumor and
36:52this is bivalve then you can see
36:54the the consistency of the tumor.
36:56You can see here that the PDX tumor
36:58actually expresses ALC and maintains the
37:00mean of phenotype of the native and the
37:02similarities between the Histology of
37:04the PDX ALC and the original patient.
37:08So this was originally we did this with
37:10the with a case that we captured through
37:13our pipeline and then we consent to
37:16the patient who agreed to only do this
37:17if we if we named the mask after him,
37:19which we did.
37:21So our approach was to implant the tumor
37:26and we we use ultrasound to actually
37:28check for engrassmans and then we we
37:30check at a second time point to to
37:32assess for growth and then we sort of
37:35split the mice between vehicle and latinum.
37:37So the question is we know that chrosotinum,
37:39we knew that Chrosotinum wasn't working,
37:41could second and third generation be helpful
37:44and so that's what we initially did so.
37:47So what we found,
37:48so this is ultrasound imaging and
37:49it's it's hard to tell but this is
37:51all tumor and this is all tumor.
37:53So there was large tumors at the
37:55time when we started therapy and you
37:57can see within within a week this on
38:00the right this is responsive therapy
38:02with most of this is like fibrotic
38:04tissue and on the left that's the
38:07disease and so on the right here
38:09you can see sort of the difference.
38:11So at day zero this is the time of
38:13therapy and you can see the ones
38:15that didn't get therapy.
38:16Grew,
38:17continued to grow.
38:18The ones that got therapy responded
38:20well and and and you can see by
38:22Western that the activity is,
38:24is is functional in terms on the access
38:27in terms of eliminating phosphorylation
38:30of ALK and some downstream signaling.
38:33And so then we went back to look
38:36at chrysatinib versus latinib and
38:38interestingly we found that yes
38:40in comparison to the vehicle which
38:42could grew after after therapy
38:44chrysatnib did have this little.
38:46Temporary stalling,
38:47but then grew out afterwards and
38:50then the Latin showed growth.
38:52So we're excited that the model
38:55was sort of mimicking what we were
38:57seeing in patients.
38:59We also tried a lectin A,
39:01which is another ALK inhibitor
39:04and found similar effect.
39:06So this is Jake Soumerai,
39:08who I work closely with as
39:10a clinical colleague.
39:11From our data we then he reached out
39:14to colleagues at different institutions
39:16who happened to have patients without
39:18B cell lymphomas for patients and
39:21they all had the ones that we could
39:24had the classerin alk fusion,
39:26many of them had been on prior therapies,
39:28a couple of them had been
39:30not responsive resotinip.
39:32So we put them on this therapy.
39:36So the one of the patients actually.
39:41Respond responded and is now in that
39:47at this point almost three years in
39:51clinical remission on the right you
39:53can sort of see what the patient
39:55initial disease and sort of after we
40:00had one patient who you know that the
40:03initial actually I should say the
40:04initial patient that we made the PDX
40:07out of responded and then recurred.
40:11And basically the transplants could
40:14not be performed in time and he passed.
40:17But since then the team has sort of
40:19realized the importance of getting
40:21the transplant done up front.
40:23So in summary, one of the patients,
40:25it's three years out and there's
40:27another young patient who was
40:29transferred to us who's now about
40:33one one year out in clinical remission,
40:36one of the patients had a partial remission.
40:39So from this we've initiated a small
40:45clinical trial to to get more cases
40:48and generate more PDX models to
40:50study this and other agents further
40:53and to also begin to look do some
40:56functional studies on on these cases.
40:58Interestingly I also really fascinated
41:00by class of plastic lymphoma
41:02because they were discovered.
41:04Around the same time in actually 1997,
41:06they, they have,
41:08they both have plasma blocks morphology,
41:11they have a very similar expression
41:13profile with the difference being the
41:15ALC because they're driven by ALC.
41:17The ALC largely cell phones
41:18drive by ALC fusions,
41:20the the plasma blocks forms are
41:21driven by Epstein Barr virus.
41:22So but I actually think that the
41:24biologies are probably pretty similar.
41:26There have been some evidence of
41:28the similar downstream signaling
41:30pathways being involved.
41:31So we we've started to view these
41:34as class obelastic type of fomas
41:36that have that both have sort of
41:39clinical clinically disciplined
41:40prognosis and no clinical trials.
41:42And we've actually set up models
41:45of these of class obelastic FOMA as
41:47well to begin to understand the the
41:50biology and develop new targets.
41:52And if you think about B cell develop
41:54B cell development starting with
41:56the Pro B cell and the bone marrow
41:58going into mature B cell.
42:01And then before you get to plasma
42:03cell where myelomas are derived
42:05from most of our mature B cell
42:08lymphomas are coming from you know
42:09the more mature B cell phenotype.
42:11There is a a very transient plastoblast
42:13phase where probably the plastoblast
42:15lymphomas and the up large B cell
42:18lymphomas arise and it it may be
42:20because these are so transient
42:22why these are are less common.
42:24But a very interested in sort of
42:26characterizing these in our models
42:27and understanding biology and
42:29coming up with new targets.
42:30Some of the questions we'd like to answer
42:33are what mediates these crozotinib
42:35failures in out large B cell lymphoma.
42:38We think that it may have to do with
42:42P glycoprotein where we know that
42:44crozotinib may be a a target for that.
42:48And then also what mediates resistance
42:51downstream after some time with
42:54the with the second and third
42:56generation OP inhibitors.
42:57And and it's interesting,
42:59I think it's fascinating that the
43:01ALK fusions actually are actually
43:03in part a better prognosis in the
43:05anaplastic large cell infomas better
43:06than the ones that don't have the ALK,
43:09whereas in the B cell infomas,
43:10it's in parts of dismal prognosis.
43:12So we're beginning to look
43:15at trying to understand.
43:17Whether those differences are different
43:19by the differences in in the fusion
43:21partners versus whether it's the native
43:23environment of B cell versus T cells.
43:24We have some experiments there to to
43:28look at that and and again trying to
43:31understand the silicon pathways that
43:33may be in parallel to plasma blast
43:35lymphomas to begin to develop some
43:36targeted therapies for that disease
43:38which there are none at the time.
43:41So for this part we you know we we basically.
43:45I found the the use of the second,
43:48the high the next generation of
43:50inhibitors in this disease and
43:52illustrate the potential of PDX
43:55models to inform therapeutic options
43:57for patients with REMALIGNANCIES.
44:03And you know more efforts are
44:05currently needed to look at adding
44:07electinim to low Latin to first line.
44:09So currently the NCCN guidelines
44:11have been modified to include.
44:13This as a go to after failure
44:16with first line therapies,
44:17but we're looking to begin to look at
44:20setting up a trial where these are
44:22included as part of the first line therapy.
44:25And then we've set up multiple
44:27lines of these at large B cell
44:29infomas and a plast cell infomas of
44:31Plast blast infomas to begin to do
44:33some of these functional studies.
44:34So I won't go through in detail
44:36with this last part,
44:38but one of the issues with lymphoma is
44:40that it's not a sheet of neoplastic cells,
44:42but there is a lot of microenvironmental
44:44cells that are actively interacting
44:46with the neoplastic cells.
44:47We know the landscape very well of
44:49the perform as I described initially,
44:51but we still can't explain the
44:53heterogeneity of the disease and the
44:55one thing that we can at this point
44:57know that is important is if patients.
45:00For this the proportion of patients
45:02that need therapy if they are,
45:04if they progress or relapse within
45:06two unit therapy they do really
45:08poorly and others do really well.
45:10But trying to find biomarkers earlier
45:12on to produce that is important.
45:14We think that understanding some elements
45:16of the micro environment may be important.
45:18So we begin to set up,
45:21we've we've set up models of focal
45:23fellow which are much harder than some of
45:25the aggressive lymphomas to begin to look at.
45:27The impact of genetic alterations in in
45:29the context of the microenvironment and
45:31what the interplay may be between the
45:33neoplastic cells and the microenvironment
45:37and the barriers again are having
45:39models for this and I'll just show
45:41you some of the pictures of the full
45:43and final model we've generated.
45:44So again this is a image
45:46of the tumor on the kidney.
45:49It's much more well circumscribed than
45:52some of the aggressive models here
45:53you can see that the cells are small,
45:55they look more like centrosized but.
45:57Mixed mixed morphology,
45:58one of the exciting things is that when
46:01you do when we look at the cells they
46:04include both B cells and the human T cells.
46:07So they they those two are present
46:09in the the tumor giving us an
46:11opportunity to begin to under to
46:13look at their interactions in vivo.
46:15This is just the immune chemistry showing
46:18the B cells and and T cells in the.
46:22In the tissue we do interestingly get
46:24some plasma acidic differentiation
46:26which you don't typically get in
46:28folk lymphoma but occurs in these
46:30models and we do get,
46:31we do get CD21 meshworks in this
46:33model although they don't persist
46:35and we believe that's because
46:37they're non non hematopoietic cells.
46:39So they persist for a period
46:40of time and go away.
46:41We we do get in NSG mice which don't
46:45have lymph nodes by definition.
46:47When we do the,
46:48when we do the PDX as we begin
46:50to see the generation of lymph
46:52nodes which is really interesting.
46:53So these these cells are tracking
46:56and into some residual primordial
46:58structure and generating lymph nodes
47:00and this just shows the B cells and
47:03the T cells within that and you do see
47:05some of this plasma state differentiation.
47:09We we do imaging to to to look at these.
47:12So this is normal kidney on
47:14the left and on the right.
47:19We'll see
47:24this, this dark thing is the tumor
47:26and we we developed this because
47:28unlike the large aggressive tumors
47:30where you can clinically sense when
47:32when the when the tumor is there,
47:35these are indolent.
47:36So we need some imaging to be able
47:38to detect engraftment and we're now
47:41working at colleagues at the road.
47:44Wignesh Shanmugam is close colleague.
47:47I'm working on spatial transcriptomics
47:49to begin to ask questions around the
47:52microenvironment and and the spatial
47:55relationships between the neoplastic
47:56cells and different subsets of the
47:59microenvironments and how that plays both
48:01in real patients but ultimately in the
48:04PD X's where we can do some manipulation.
48:08So I just want to make sure
48:09I don't out of time.
48:10So yeah, so in conclusion here we.
48:14We are,
48:16we are using you know paired human
48:19focal FOMA and focal PDX samples that
48:22characterize the the microenvironment,
48:24the clonal populations and to explore
48:26their nature and roll the crosstalk
48:29between these cell populations.
48:30And we anticipate that the INTRIVIAL
48:32model will serve as an ideal system to
48:35really interrogate focal formal biology
48:37and understand the mechanism mechanisms
48:39of targeted therapies and resistance.
48:43So with that I want to thank you for,
48:47for coming in person and
48:49for those who are on zoom,
48:51thank you for for listening
48:53and I'll take any questions.
49:03Very nice.
49:05Two questions Chris really like getting.
49:14It's
49:30about is now we know that uniquely
49:35starts by in that general center.
49:44Yeah, I mean presumably like the
49:47potentially the map kinase might
49:49be driving proliferation in those,
49:51that's one possibility and
49:53you know there have been
49:58you know people looking at micro
50:00RNAs and other other downstream.
50:03But we don't have a clear clearly that
50:05not it's not the same mechanism as
50:07having BCL two over express but that
50:09also may may be part of the reason why
50:11we never see systemic involvement by we
50:13don't see you know long term systemic
50:16involvement by these pediatric type.
50:17They're pretty limited and the you
50:19know to the point where there was
50:21a lot of debate about whether these
50:23should even be called lymphoma, right.
50:33Maybe I wonder where it's in
50:38on the OR the machinery. Yeah,
50:43yeah. There's no direct. Yeah.
50:45Second question I wanted to
50:46ask is on the latter app,
50:48the PDX is really pretty exciting
50:51to be able to use those.
50:54But you brought up from the end that
50:56you also want to look at the micro.
50:59Yes, you do get to. Have some of it
51:02in the future planted,
51:04but I think a lot of you guys,
51:05some of the issues are that for
51:09multiple passages that sort of
51:11leave that and you start winding up.
51:13Yes, yes. So, so I'm wondering if
51:16a lot of you guys explore which
51:20types of humanized type models.
51:24We're we're starting to to to
51:26look at the humanized models
51:27we've had it's a little bit,
51:29they're a little bit tricky to to
51:30use and they're extremely expensive.
51:32So you have to like you have to temper
51:34how you do things get this jump into it.
51:36And then we also in terms of match and
51:39we've tried to attempt to in our bank,
51:41we actually can prospectively
51:42collect blood from the patients who
51:45consent and so we've actually tried
51:47to think about matching those which
51:48takes some time and then you have to
51:50you know so we're working on that.
51:52It's a much harder experiment.
51:54Yeah. And you're right.
51:55Over time, it's a, you know,
51:57we do run into, you know,
51:58you lose like environment and how.
51:59Yeah.
52:01Thank you.
52:05Hi, can I ask you a question?
52:06This is Jeff Sklar on Zoom
52:11A2 part question. You know, I wonder
52:15about selection that's going on.
52:23Murders once they're established
52:25and compare the sequences of the
52:31yeah, we we do do that,
52:32we we do sequence them and do RN A/C
52:37to to compare and the the mutations
52:41that are present in the patient
52:43are typically present in the,
52:44although there are some.
52:47They're not 100% but they're
52:49pretty similar the the main
52:50chromatin modifying gene
52:57and so one of the points I was
53:00kind of getting at is do you ever
53:03see transformation to large cell
53:05in the rafted folliculars? Yes,
53:08we have. We have seen that in a in a few
53:11cases we can't get it to recurrently occur.
53:14So we'll have it as a.
53:15As an event and you know we're excited
53:17about the possibility of leveraging
53:19that to understand transformation,
53:21but it's it's hard to get as a
53:24recurrent issue as a you know,
53:26as a model of that transformation. Thanks.
53:38Yeah,
53:42very.
53:52Yes, yeah. Yes. Yeah,
53:57yeah,
54:02yeah. And that's also
54:03in my bucket in my mind.
54:05But I never really talked
54:06about it because no one knows,
54:07you know, it's so rare.
54:09But I really think that
54:10ultimately we're going to find
54:12that these have very similar.
54:14Analogy and if targets and in one,
54:18then my other question
54:28what are your thoughts really
54:30about this grading versus not.
54:35Yeah. So to be honest when
54:38I when I first joined
54:40the committee and having trained.
54:43With Nancy Harris.
54:44And I got onto the committee
54:46and the committee was like,
54:47we need to get rid of this grading.
54:49And my eyes were like,
54:52yeah, because that was like,
54:54yeah, I was like, wow.
54:56And I found myself defending really trying
54:58to defend the the point of grading.
55:00But then they made me see that, you know,
55:03with some of the newer targeted therapies
55:05that are being used, there is no.
55:08There's no difference really in in the
55:11patients that are 3A versus 1 to 2.
55:12And then the other thing is like
55:14I mentioned the POD24 where where
55:16patients that relapse or occur
55:18within two years they do much worse.
55:20And then the patients who don't,
55:22if you look at some of those papers
55:24they they they show you like in the
55:26supplements like the the the breakdown of
55:28these cases and there's an even number
55:30of grade one to two grade 3A in those.
55:32So it's actually not predictive of that.
55:34So I actually think there's a pretty
55:37good argument of not doing grading.
55:40I think the consensus of 3B is
55:44really more like DLBCL essentially,
55:45which is basically sheets of large cells.
55:47This happens to have follicles.
55:49So what the decision was was
55:51we would lose it.
55:52But usually when you think about
55:54making a change in a different way,
55:56show there's sort of this unwritten
55:57rule that you have to have data.
55:59To support it and interestingly for that,
56:00there is no data to talk
56:02about removing something,
56:03but it's almost like when you look at the
56:05origins of grading which was a little bit,
56:08there's not much data there.
56:10So it's and then the idea was
56:11like over time we'll figure it
56:12out but it never got figured out.
56:14So it's almost removing something
56:15that was never.
56:16So I think that's the,
56:17well what we did do is was
56:20suggest that grading be allowed.
56:22So it's,
56:23it's not required but at local
56:25institutions like ours where some of the
56:27oncologists really want the grading.
56:28We still have the grading,
56:29but we just wanted,
56:30I think the idea was to make it
56:31clear that it wasn't required.
56:32And I think that sort of the
56:35balance that we left were left with.
56:40Yeah, right, right.
56:45Thank you for the question.
56:55Are you looking for the DA sequence?
56:57Are you looking for the most of PCM who
57:00might support the transportation but
57:03have you snapped any translocation that.
57:08No. So there's no, there's
57:10usually no rearrangement.
57:11Igh would be the common partner,
57:16yeah no I I don't know of any.
57:18I know that there's some new
57:20technologies that are that can
57:21that can pick up some uncommon.
57:23Types of transvocations and we've thought
57:25about submitting some of these to that,
57:26but there's no evidence to date of any
57:29rearrangements in these in these lesions.
57:33So the second question regarding more
57:36general for the P7 coma, so really P7
57:41coma that have ITG and location is.
57:43So what is driving by the ITG from overreach?
57:49Right. So because the right now the
57:51uncle gene always replaced by the ITG
57:54which so it's high and expressed.
57:56So my thought is that we if we instead
58:01of becoming even the uncle gene which
58:04is coming out the ITG transporting
58:06the level you keep the water
58:10transportation uncle gene fashion low.
58:12You see that will be high in your strategy.
58:15So targeting, targeting the
58:17rearrangement you're saying.
58:18No talking even though globally
58:21and trying to reliable because
58:24this this model actually friendly
58:26controlled by the ITG reliable.
58:30Yeah no, I've never really thought
58:32thought about that interesting idea.
58:38Thank you.