Pathology Grand Rounds: November 10, 2022

November 15, 2022

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Cytology Samples and Pre-analytic Processing for Molecular Testing (Cytological Samples for Biomarker Analysis: Usefulness, Advantages and Limitations) presented by Maria Dolores Lozano, MD.

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00:00Good afternoon and.
00:03For everyone here and over the zoom.
00:09Welcome to the pathology ground round.
00:13Is my great?
00:15Honours and privileges to introduce
00:19today's grand rounds speaker,
00:21doctor Lazano.
00:24Whatever Asano complete completed
00:27for medical education in the early 90.
00:32In the University of Navarra, Spain.
00:37The university is located in the northern
00:40Spain in the city of the Pamplona.
00:45Which is the famous for
00:48Annual full fighting festivals.
00:52And it is such a beautiful city and
00:56you can appreciate through Doctor
00:59Lozano's many social media posts.
01:03And it's things Doctor Rodano
01:05likes the city so much so
01:08she never left the city.
01:11So she is currently
01:13a professor of pathology and the
01:15chair of the Anatomic Pathology in the
01:19university at the University of Navarra.
01:23Daughter Rosanna is renowned sociologist
01:27with expertise in cytopathology,
01:30molecular pathology and. Thoracic pathology.
01:36She is a current president of the
01:40Spanish Society of Science.
01:48Doctor Rodano has been invited to
01:51give presentation at pathology meetings.
01:54And seminars and tutorials worldwide.
02:01And this morning we have great unknown
02:05session with the resident and fellows.
02:10Daughter Rosanna has been recognized as
02:14an expert in using psychological
02:17specimens for the N 3 testing like
02:21molecular test and biomarker test.
02:24And which is the important for?
02:30Titering uh therapy for
02:33patient with lung cancer.
02:36She has published more than
02:38100 peer review articles,
02:40reviews and editorials in the
02:45passage and Medical College journals.
02:49As we know,
02:50Cytologic specimens are the important for
02:54diagnosis and tumor classifications.
02:57And does he have sometimes the only specimen
03:02available for diagnosis diagnostic workup?
03:06However, we face this challenge in using
03:10psychological specimen for entry tests.
03:13And due to inherent issues of psychology
03:17specimen including low cellularity,
03:20low tumor burden,
03:23and variable cytological preparations.
03:27So we hope today's ground run by Doctor
03:31Rodano will help us learn the best
03:35approach how to tackle these issues.
03:39Without the further ado.
03:40I would like to give the floor
03:43to doctor Rodano.
03:44That's Romano.
03:45Face.
03:51Well, thank you so much doctor
03:53Kai for your kind invitation.
03:55It's a absolutely a privilege and
03:57an honor for me to be in here.
04:00And I'm happy and I'm very
04:02thankful to all of you,
04:03especially Doctor Kai and Doctor Levy.
04:06Levy. So I want to just start with my dog.
04:10Probably you, most of you or many of you
04:13know more than me about all this world.
04:16But I'm gonna tell you something
04:19based on mainly on. Experience.
04:21So let's start with my talk and.
04:26OK, I don't have any conflict of interest.
04:31So this talk is going to be focused
04:34especially in into two parts.
04:36So first one I I want to give a practical
04:39overview of the types of psychological
04:41samples and the ways to obtain them
04:44and to manage them for biomarker analysis.
04:46And the second part of the talk is
04:49going to be a around the possibilities,
04:52advantage and limitations of
04:54psychology for all the techniques.
04:56What do you use for biomarkers,
04:58especially in the field of non small
05:01cell lung cancer and we start,
05:03I want to invite all to you,
05:06all of you to Spain.
05:07But because we are not going
05:08to Spain right now,
05:10I will not try to show many
05:12parts of my country.
05:13It's a small country but very different.
05:16This is starting for the North,
05:17this is the north and we have many
05:20fishing villages in the Northeast
05:22region is called Cantabria and
05:24it's where the Atlantic Oceans.
05:26Entered into Spain and it's
05:28called Mark Cantabrica.
05:29Cantabrica.
05:30So this is a beautiful, beautiful place.
05:32You are invited and explain there is a
05:34lot of art and this is in the north.
05:36This is Bilbao, the city of Bilbao
05:38and this is the Guggenheim Museum.
05:41The Guggenheim Museum is very
05:43poor while known museum,
05:46especially this huge doggy who is at
05:48the entrance and is made of flowers.
05:51The whole year is like this but I'm
05:54not here to talk about Spain and the.
05:57Very beautiful things that we
05:58have with Spain.
05:59I'm here to talk about.
06:02Cytopathology and biomarkers
06:03especially in the field of lung cancer,
06:07non small cell lung cancer.
06:09So and the first one,
06:10one thing that I have to share
06:13with you is that the impact of the
06:16new W WHO classifications and the
06:18advance that supposed compared to
06:20the old one that is dating from 2015.
06:24So basically the new admin say new things,
06:29some of them are not very new because
06:31the first thing they authorize.
06:32This is this one morphology first
06:35remember morphology first supported
06:37by immunohistochemistry,
06:39and then and then in this order,
06:41molecular studies.
06:42In the new classification the
06:45there is an emphasis,
06:47a great emphasis on genetic testing,
06:49and it's a section entirely
06:51dedicated to the classification
06:53of small diagnostic samples.
06:55This is important.
06:56There are more new things that are
07:00mainly dedicated to surgical pathology.
07:02But one important thing to me is that
07:05and we saw this morning in the seminar,
07:07there is a recognition of a
07:10smart C for deficient tumors,
07:13thoracic deficient tumors as a new entity.
07:16So we have to think always that
07:19cytopathology and surgical pathology
07:20are going always hand by hand.
07:23We cannot, you know,
07:24forget surgical and do only.
07:27Mythology so we we worked together
07:30and we enriched together.
07:32So this is the the script of my job.
07:35First, what is a biomarker?
07:38Then I'm going to talk just briefly
07:40about the rationale for the need of
07:43biomarkers using ancillary techniques,
07:44of course.
07:45Then I'm going to talk about
07:47the rationale for the use of
07:49psychological markers for symbols.
07:51Sorry for biomarker testing and then.
07:54And move to the future related
07:58with the samples,
07:59the pre analytics,
08:00the processing techniques advantages
08:02and limitation of different
08:04samples from different techniques.
08:06So let's start from the beginning and
08:09what happened with biomarkers in the
08:11field of pathology and cytopathology.
08:14Of course even the biomarkers
08:17have many functions.
08:19I would say we are more focused
08:22into into into advantage.
08:24Biomarkers,
08:25one is directly related with the
08:29diagnosis with biomarkers can
08:31help us to define the subtype
08:34of lung cancer so we can give a
08:38confident diagnosis using specific
08:40biomarkers and another biomarkers
08:43are predictive if this is important
08:46because they inform us and the
08:49oncologist about the treatment
08:52for this particular patient.
08:55Quickly the rationale for the need of
08:58biomarkers using ancillary Chinese
08:59what is why biomarkers because
09:01we live in the in the world of
09:03personalized medicine and if you go
09:05to the FDA definition the FDA says
09:08precision medicine is providing the
09:11right treatment at the right time,
09:14every time to the right patient.
09:16But I think they forgot one important,
09:18very very important aspect is
09:20precision medicine is providing
09:22the right diagnosis to make.
09:24Possible the rest of the sentence, right?
09:27Because with the right diagnosis we
09:29what we do is an accurate diagnosis
09:33that allows for a specific treatment.
09:36So at the end it would benefit the patient,
09:38which is our main goal.
09:41I want to represent this in
09:44this in this slide.
09:46In fairly all these patients with yellow.
09:50A few years I have the same
09:52diagnosis at the same stage non
09:54small cell lung cancer stage
09:56four same disease more or less.
09:59But if you see this red mark patients
10:02this is a 61 male smokers squamous
10:06with a curious mutations male,
10:0954 non-smoker adenocarcinoma,
10:11ALK translocation,
10:12female non-smoker adenocarcinoma,
10:14Egypt permutation etcetera.
10:16So we all agree pathologists are oncologists.
10:20These people,
10:20these patients have a different
10:22tumors and then they have to
10:24be treated in a different way.
10:26So that's their representation of the A
10:31targeted medicine and personalized medicine.
10:35Third Point of my dog is the rationale
10:37for the use of psychological
10:40samples for biomarker testing.
10:42And what is the rationale?
10:43The rationale is easy to understand
10:45especially in advanced stages.
10:47It what is the role of psychology
10:50and precision medicine.
10:51First is the the the less
10:54invasive invasive method for
10:57for getting tumor. Many times.
11:01In some cases it's the only method
11:04to get tumor for example pancreas,
11:06advanced lung tumors or thyroid or
11:09other locations importantly in no need
11:13for the calcification where the tumor
11:15is diagnosed through bone metastasis.
11:18You know that in the the calcification
11:22procedures damage the tissue and some
11:25immunos etcetera don't work and if you if
11:28if we do a good finding the last spiration.
11:31We can get a pure population,
11:33more or less pure population
11:35of neoplastic cells.
11:37And this is the scenario we face everyday,
11:40everyday, everywhere.
11:41Now there are some challenges in the
11:44diagnosis of non Marcel and cancer
11:46and also other types of cancer.
11:49There is a they they are there is a
11:51demand of for comprehensive and complex
11:54diagnosis using minimal invasive procedures.
11:56We live with that every day.
11:58We live in the world of personalized
12:00therapies and we have to integrate
12:02biomarkers in in the clinical
12:04management of the Council patient
12:06not only in the diagnosis.
12:08But also in the clinical management during
12:11the whole disease and so far is is too bad.
12:16But so far many more than 50% of non
12:19small cell lung cancer patient came to
12:21the hospital with advanced diseases.
12:23So in these patients we only have
12:26psychological or or small biopsy for
12:29making the diagnosis are all the
12:31biomarker analysis at the same times
12:33fortunately there is a development
12:35of imaging techniques and new.
12:37Details and excited at that allows for
12:40not invasive approach but we get the
12:43same type of material psychological
12:45materials and are small small biopsies.
12:48So if I need aspiration at the end
12:49and if I need an aspiration biopsy
12:51are in the first line in the diagnosis
12:54and in the management of patient
12:56with non small cell lung cancer.
12:59And remember neoadjuvant therapy is
13:01coming and it's really coming so when
13:04adjuvant therapy non small cell lung cancer.
13:07Grayson is a reality is gonna be soon.
13:10We will need to to deal with
13:12more type of these small samples,
13:14biopsies,
13:15final respiration etcetera to study
13:17the tumor and then to the native ANSI
13:21and etcetera like in pancreas for example.
13:24There are many papers in the literature
13:27that prove that these biomarkers
13:30can be tested successfully in small
13:33biopsies and in psychological samples.
13:36These are three I love.
13:37This paper published in New England
13:39Journal of Medicine in 2017 is
13:41very informative,
13:42especially for for young people,
13:44very very clear.
13:45And these are other other studies
13:48that said that biomarkers can be
13:51applied to this type of small samples.
13:55Also in 2018,
13:57the guidelines change the the big
14:00groups of pathology from the United
14:03States and very quick the the the
14:06Society of Medical Oncology join
14:09and agree with the pathologist
14:11in the sense that before in 2013
14:15only cellblock was considered.
14:18An option for biomarkers using in
14:21in psychology or small samples.
14:24But now since 2018 pathologist
14:26says that either cellblock or other
14:29psychological preparation are suitable
14:31specimens for lung cancer biomarkers
14:34molecular testing and if you look
14:36at the what the oncology community
14:39says they say pathologists may
14:41use either cell block or smears.
14:44They forgot all kind of other preparation,
14:47but I agree with that.
14:49Because he's there.
14:50The cell block and smears is the more
14:52common sample we deal with every day.
14:54So the question is that the the that
14:57any sample with adequate cellularity
15:00and preservation may be best,
15:02and this is a huge agreement about that.
15:07But what is true is that is,
15:09is it at this I feel like this
15:11probably you you feel the same
15:13that I feeling that cytopathology
15:15and land cytopathology especially
15:17is in continuous revolution.
15:19There is the need of implementation
15:21of new diagnostic tests.
15:23We face new challenge,
15:24there are new tools in the market around
15:27a new elements of necessary incorporation
15:29and all this is happened very quickly.
15:32So probably as in this
15:34paper published in 2018.
15:36And they say that probably time
15:38for a next generation pathologist.
15:41I think we have the next
15:43generation pathologist here.
15:44So it's our duty to try to to
15:48encourage them because yes,
15:51it's,
15:51it's pathology has changed for for
15:53those who yeah who are a little
15:55bit older not all but a little
15:57bit pathology has changed a lot.
16:00So we have to be ready for all the
16:02changes and to to adapt very, very quickly.
16:05So it's important subtyping
16:08non small cell lung cancer.
16:10Yes,
16:11the new Double H WHO classification said
16:13that we need to subtype nurse Marcel and
16:17cancer regardless the type of the samples.
16:19Why?
16:20Because regardless this subtyping the
16:24oncologist without any biomarker oncologist
16:28would give a different usual chemotherapy,
16:32so squamous.
16:33A tumor with the scammers
16:35features will receive a different
16:37chemotherapy that that jumars that
16:39don't have squamous features.
16:42So this is very important.
16:43So at the end morphology
16:45is the first biomarker.
16:46We cannot forget this because in the
16:48world of NGS and all these fancy
16:50techniques we can forgot and morphology
16:53still is the first biomarker it's
16:55important for especially for young people.
16:57But not always is is easy.
17:00We saw many cases this morning because
17:04who is able to subtype these three cases?
17:08It's impossible,
17:09almost impossible.
17:10So the recommendation of the big
17:13societies is to reduce the diagnosis
17:15of non small cell lung cancer and OS to
17:18the low level possible less than 10%.
17:20So we need help and we need and
17:24we need help and we need help.
17:27It is very, very well.
17:28Explain I think I love this
17:31paper is published.
17:32And some months ago in June 2020.
17:37It's a it's a working group.
17:40It's a survey that you 2
17:42dinner determine specific sites
17:44for motorcycle morphology.
17:45Sorry criteria for site type,
17:48nonsocial and concept into adeno or squamous.
17:51This paper they they involves
17:55313 experts cytopathologist and
17:58they should satisfy 1.
18:03119 non small cell lung cancer.
18:06This expert group of pathology selected
18:0923 predefined cycle morphological feature
18:12to classify and then data were analyzed
18:16using machine learning algorithms
18:19develop so to to see what is happening.
18:23OK the results are here the concordant
18:27cytology results or succeed in close to 54%.
18:32Of the cases.
18:34Misclassification rates were higher in
18:37lung squamous cell carcinoma and the
18:41important point was keratinization.
18:43Keratinization when when present
18:45recognized squamous cell carcinoma.
18:47Very easy. Everybody agrees with that.
18:51But in absence of Keratinization,
18:54the machine learning algorithms
18:56developed on the basis of expert
18:59choices were unable to reduce
19:02psychology and a waste diagnosis.
19:04Rate without increasing
19:06misclassification rates.
19:08So what I mean what I wanna say that in
19:11the world of artificial intelligence,
19:14please don't forget human intelligence,
19:17human intelligence and
19:18artificial intelligence,
19:19how to go hand by hand because one
19:23benefit from the other and the other
19:26benefit for from the from from what?
19:28So that's very important.
19:30So the conclusion of this,
19:32of this study is that.
19:34It says suboptimal recognition as cell
19:38carcinoma in absence of keratinization.
19:40So we need help at the first.
19:42Help we need is the immunohistochemistry
19:46immunocytochemistry in pathology.
19:48Especially in pathology banning all
19:51cancer first is used for diagnosis
19:54in case of non small cell lung
19:58cancer to subtyping this tumor.
20:00Second to to find out the primary
20:03tumor in case of metastasis.
20:05And then for biomarkers,
20:07you remember this picture,
20:08city one is positive so it favors
20:11adenocarcinoma and the panel of
20:14immunohistochemistry of antibodies
20:15in lung cancer is very simple,
20:18very easy TD1 cytokeratin 7 and
20:21naps in Paladino carcinoma and
20:24P40P63 for squamous cell carcinoma.
20:26But don't forget these two new not really
20:29new antibodies smart support efficient
20:31tumors are not one positive tumors.
20:34So we had to.
20:36Keep in mind these two possibilities,
20:38as we saw this morning in the in the.
20:40In decision.
20:41So yes to remain that precision
20:44medicine starts with an
20:47accurate morphological defense.
20:48Second utility of of immunohistochemistry is
20:51to determine the origin of metastatic tumors.
20:55This is a real case,
20:56sorry because this is in Spanish.
20:58It does is pericardial fluid.
21:00We don't know anything,
21:02anything,
21:02nothing, nothing,
21:03nothing I think is that happened everywhere
21:06in the world is pericardial fluid and
21:08this is the the pericardial fluid is.
21:11Tumor of course is an adenocarcinoma.
21:13We go to the clinical.
21:15We went to the clinical records
21:16of the patient.
21:17We we knew that the patient had a lung mass,
21:19so we did.
21:21T1T1 is positive.
21:22We confirm that the origin of
21:25this pericardial effusion is
21:27the adenocarcinoma of the lung.
21:29I know the real case.
21:31This is a very young lady,
21:3232,
21:33female only smoker with very
21:35disseminating disease of unknown origin.
21:38We did a direct financial aspiration
21:40of our supraclavicular live now
21:43and if if we didn't have know that
21:45the basement have a landmass,
21:47we were thinking in.
21:49A gastric tumor or even breast right,
21:52but if one was positive so
21:55the the origin of
21:56this tumor is slack but besides of
21:59that is mandatory the integration of
22:02biomarkers in the clinical management
22:04of lung cancer patient and as said
22:06before so this is nice to have this
22:09we know that this the the last case
22:12only the pericardial fluid is origin
22:14in adenocarcinoma of the lung but
22:16this patient have a mutation of EGFR.
22:19So it's a simple way to do
22:22precision medicine.
22:22The other lady is is nice,
22:25is better to know that the lady have
22:28an alt translocation to support
22:30patients can go to a specific therapy.
22:34So is that we call day-to-day routine
22:37precision medicine based on psychological
22:39samples without any complication,
22:42but very helpful for the patients, OK.
22:44And I can't resist to show you these cases.
22:49These cases motivate a lot to me.
22:51That case is dates from 2009 and I really
22:56discovered the value that psychopathologies
22:59have in in the in in this world.
23:02Let me let me show you.
23:04This is this lady came from a second
23:07opinion from another city in Spain is
23:09a female is 72 years old at that time.
23:12non-smoker disseminate disease.
23:13Look at the pet even brain.
23:15That's disease,
23:16** *** has been only offered
23:19a palliative treatment,
23:21so she decided to go to another
23:23institution and we perform a
23:25direct financial aspiration of
23:27this note and it's easy to to know
23:30that this is an adenocarcinoma.
23:32Is the one nuclear cytokeratin cytokeratin
23:367 cytoplasmatic act even the patient
23:39have a deletion of exon 19 and 85 so.
23:44Theme so three months later with TK I
23:48is inhibitor the patient was like that.
23:51So to me it was like a before and after no.
23:55So it's it's important with us so
23:58simple technique to to be to be able
24:01to help people like this like so.
24:03But it's important to do molecular and
24:06to do to inform about biomarkers of
24:09course is mandatory and there are the
24:12masks the shores the mask you know.
24:14Everybody knows you get park and
24:18grass and exon skipping next of 14
24:21and met Ralph Al Rose and Jack Red.
24:25Of course PDL one and metabolic fication
24:27is coming into this group also.
24:30And all this happened in adenocarcinomas
24:33and squamous of course PDL one and
24:36now in track is getting with is
24:38indeed into the room.
24:39But if if the patient with schemas
24:42Histology is not a light smoker.
24:45Or is younger than 50 goes through the
24:49adenocarcinoma site in terms of biomarkers?
24:52Unless moved to another part of my talk,
24:56but before I went to stop in Barcelona and
24:59I want to stop in the Sagrada Familia.
25:02Some of you know but Sagrada Familia
25:05is impressive from outside but I can
25:08tell you to me is more impressive from
25:10the inside. It's absolutely gorgeous.
25:13So you have the the opportunity I
25:18I would like to invite you to see
25:21this wonderful so the the third,
25:22the 4th part of my talk is yes,
25:25the management and the advantages
25:26and limitation of the psychological
25:28samples for biomarkers.
25:30I want to make a stop and this is Valencia.
25:33Valencia, little bit S in the
25:35Mediterranean coast and Valencia.
25:37This is called the City of Art
25:40and Science and it's wonderful.
25:42Valencia is a very nice city,
25:44so you are welcome also.
25:47So to talk about this,
25:48we have to ask us,
25:50or at least I I make myself, two questions.
25:54How to ensure adequate diagnosis
25:57using minimally invasive methods
25:59and therefore using small samples.
26:02And the most important question,
26:03how to provide all patients,
26:06all everyone with the benefit
26:09of effective therapies.
26:11So with adequate samples,
26:13with inadequate management of these
26:15samples to be able to do anything we need
26:18and something that is very helpful it grows.
26:20I want to stop in this part because
26:23we all know the value of rows.
26:26Rows is I would say mandatory
26:30especially for two things for.
26:32To to take care of the Preanalytical control
26:36and to make a proper triage of the sample.
26:40This is a paper that I like a lot
26:42published in 2019 talking about the role
26:45of site to check in in the in the roles.
26:48Obviously they do a great job,
26:50but I love this.
26:52And we need a standardized protocol for
26:55tissue management and for psychological
26:57management and the this is very important
27:01in the era of personalized medicine,
27:04adequate,
27:04should they not sufficient tissue for
27:07diagnosis and for molecular are ancillary
27:10tests not only sufficient cells or diagnosis,
27:13but also it's mandatory to do
27:16molecular and ancillary tests.
27:17But one thing that we really is
27:21a limitation of psychological.
27:23Numbers is the lack of architecture and this
27:26is true it's a limitation even cell blocks,
27:29regular cell blocks block architecture.
27:32OK so my my,
27:33my my question is what do you think?
27:36Can we preserve the architecture of the
27:38samples doing fine needle aspiration?
27:41Yes, no, it depends how how you,
27:44how we approach the yes or the no.
27:47But they will say maybe,
27:49maybe why not,
27:50let's give the doubt a chance and why
27:53because we are all the time using the
27:56the term NGS next generation sequencing.
28:00We can add two more terms NGN
28:03like generation needles and NGP
28:05NEXT generation procedures.
28:07In the market you know there are
28:10a huge development on new needles
28:13that allow to directly obtain thin
28:16film Anders regardless of the needle
28:20G and this is a real examples.
28:23And there are another technique that
28:25is called next generation procedures,
28:27although it's not at all new
28:29because it's published in 2015,
28:31is called wet suction technique.
28:34That and the the only thing that
28:36they do is just to put a little bit
28:40of physiologic in the syringer to
28:42increase the pressure of aspiration.
28:45So we get these.
28:46These are real example from institution.
28:48We get dieted cell blocks,
28:51dieted thin, thin cylinders.
28:53So the architecture is preserved.
28:56What is what is important is
28:58to introduce very the sample
29:00very very very very slowly,
29:02informally,
29:02because as as atheist is very thing it fixes
29:06at the end. We got like a very thin
29:09cylinder with well preserved architecture,
29:12cells, cell block we put in formally
29:15in paraffin and we want the and
29:17we can do whatever we want to do.
29:20So that techniques, both needles
29:22and procedure have many advantages.
29:25We we can have a psychologist.
29:27Logical correlation like in this
29:28case we can choose the most
29:30appropriate sample for each technique.
29:32For example, with a good cell blocks we can
29:34do without any problem any many antibodies,
29:37immunohistochemistry and in in
29:39particular in our institution we
29:42prefer to use smears to do fits.
29:44Many pacing can benefit for clinical trials.
29:47You know cellblock,
29:48regular cell blocks and smear don't allow
29:51this patient to go to clinical trials,
29:54but these are biopsies and
29:56some clinical trials.
29:58But yeah, yeah,
29:59they can accept these type of samples
30:02because at the end this small biopsy.
30:05And we can do many molecular analysis
30:07of course and even we can do studies
30:10of the immune microenvironment
30:11with these nowadays very important,
30:14this is a a schematic representation of
30:16the different type of samples we can get.
30:18This is a very,
30:20very recent paper published
30:22with Fernando Smith,
30:24actually is published in September.
30:26And this is an algorithm just to remind
30:29you that if we have enough material to do
30:32a morphological diagnosis and biomarkers.
30:35Everything is OK because we can
30:37go and do anything.
30:38They must biomarkers that we have to do.
30:42But what happened if we don't have
30:45enough material to we have only
30:48material to do morphological diagnosis,
30:50but we cannot repeat the procedure
30:53to get material for biomarkers.
30:55So we have to take,
30:57we have to be in and mind to we
30:59have to keep in mind that this
31:02is a incomplete diagnosis.
31:03Morphology is good, but this.
31:05Nowadays is an incomplete diagnosis,
31:08OK.
31:10So yes practical issue about
31:12Minister chemistry considered an
31:14also PDL one fees NGS yes some words
31:18about tumor microenvironment you
31:20thinking So what are the the pre
31:22analytical factors important for
31:24the minister chemistry or or know
31:26very well the important message of
31:28this slide is this one cell blocks
31:30should be fixed informally none in
31:32other fixative just informally what
31:35happened if we don't have Sylvia.
31:37The basin has not option to anything.
31:41I think the patient should have any option.
31:44So it's a way that swimming
31:47against the flow when we try to
31:49do immunohistochemistry on smears.
31:51There is a lot of contradictory results
31:54in the literature because mainly
31:57because there is a lack of optimization
31:59of immunohistochemistry protocols
32:01for psychology slides and there is
32:04a lack of quality controls and validation.
32:07This is the main problems when
32:09you we use non cell block samples
32:13for immunohistochemistry.
32:14This is a table comparing based on
32:17the publication in the literature.
32:19Cellblock Papanicolaou stain
32:20at the smear and stain a deep,
32:22quick and liquid base with the results
32:25and if we consider it non cell block,
32:28the best results are obtained using
32:31performing ministry chemistry on
32:33Papanicolaou stained smears.
32:35And the wars only with base preparation
32:39because the fixative is methanol.
32:42And as we as we all know methanol
32:45give some problems, blocks,
32:47some antibodies and many other things.
32:50But the the the truth is that psychological
32:53specimen non cell blocks are under used
32:56for predictive immunohistochemistry.
32:58There are practical difficulties and
33:00lack of standardization for performing
33:03ministry chemistry on null cell blocks.
33:05Provincial preparation and we need
33:08validation, we need controls,
33:09we need protocols to be able to get
33:12good results and made them these
33:15results comparable to celebra.
33:17So we have a lot to do.
33:19Regarding the Minister commissary,
33:23practically minister chemistry,
33:24these are the pre analytical in psychology.
33:27These are the pre analytical
33:28factors we have to take care of
33:30fixatives type of preparation,
33:32psychological preparation,
33:33stain, coverless, cover,
33:35sleeping and antigen retrieval.
33:36And this is simply summarize our experience.
33:40So if we do perform in Ministry,
33:42Commission, cellblock it's easy,
33:45it's a formal in fixation in this
33:48abnormal paraffin OK there is no problem.
33:50But what happened if we are trying?
33:52We don't have cell block and we want to
33:55give some information for this particular
33:57patient and we only have smears.
33:59We do personally we do over
34:02Papanicolaou stained desmarias we
34:04use in our Department of Plastic
34:07Coverslip is is good because you can
34:10take it out in 2 minutes in acetone.
34:13So it's very easy to get out
34:15and the the cells are perfect.
34:17And what about on teaching retrieval?
34:19Many. It probably you don't remember.
34:22Of course I hope you remember,
34:24but in case you don't remember alcohol
34:28based fixatives or by causing tissue
34:31dehydration and protein violation.
34:34So this is very gentle with the cells so
34:37the majority of antigens do not require.
34:41Any form of antigen retrieval or
34:44this antigen retrieval has to be
34:47modified and be gentle I would say
34:49because the type the intrinsic type of
34:52fixation of alcohol based fixatives.
34:56What would we have to do?
34:57When do we do anything? Fees.
35:00Molecular or immuno on smears.
35:03We have to immortalize in some way,
35:06in some way in the the preparation
35:08because we we will lose them.
35:10We have problems, even medical,
35:12legal problems.
35:13So what we can take pictures or we can scan,
35:16we scan the preparation.
35:17This is a plural, a pericardial fluid,
35:19sorry,
35:20from a patient with squamous cell carcinoma,
35:22the only group of cells in the
35:24in all these smears.
35:25Of course this one,
35:26it's hard to say this is a squamous
35:29cell carcinoma only by morphology
35:30at least to me it's very hard.
35:32But we we perform a P40,
35:35we scan the preparation and P40 same group.
35:38So it's positive.
35:39So it's a, it's a very,
35:42very good help in this case.
35:45Let's move to to talk about a little bit
35:48about biomarkers for immune checkpoint
35:50inhibitors and the star is PDL one,
35:54but so far immunohistochemistry.
35:56This only the available biomarker we
36:00have to determine the the positivity
36:03of negativity of PD one in.
36:06In for, for, in for decision a
36:09clinical decision on immunotherapy.
36:11So, so as they they sources pathologists
36:15have been challenged to perform many
36:18says with a very limited sample.
36:20I recommend to you these two papers,
36:22one is published by our group last year
36:24and the second one is published this year.
36:26But the group of Pantano and
36:28there is a good review of PDL one
36:31and and ministry chemistry and
36:33PDL one and cytological samples.
36:36These papers talk about briefly,
36:39I'm gonna just mention pre
36:41analytical consideration.
36:42Remember cell block speaks in formalin
36:45and smears in alcohol and 96.
36:48Analytical consideration,
36:49control, sample adequacy,
36:52interpretation, clinical correlation,
36:55immortalizes smear, physical pathology,
36:59etcetera.
37:00Positive staying positive stain is
37:02considered when there is a complete
37:04or partial membranous staining
37:06irrespective of the intensity.
37:08So this is a smear with it's easy to
37:13interpret because it's membranous.
37:15Beautiful membranous staining,
37:17negative stain,
37:19negative stain for PDL one is when
37:22we don't see this membrane staining.
37:26Ohh when we see this kind of granular
37:29or even nuclear staining anything
37:31that is not membrane is negative.
37:34OK, even in this case that seems to be,
37:37but now it's a granular cytoplasmic
37:39staining which is considered
37:40negative pitfalls.
37:41We have to avoid macrophages and all
37:46other inflammatory cells that can
37:48mimic to and can pretend to be a tumor cells.
37:52Sometimes macrophages are easy,
37:54but sometimes macrophages also.
37:56Staying at the membrane so this is
37:58this is a problem sometimes and
38:01these authors also talk about post
38:03analytical considerations such a
38:05correlation with clinical outcomes
38:07and stress the importance of external
38:11quality controls and and validation
38:14because we know that there is a draw
38:17a drug behind all these all these tests.
38:21So when we talk about Cellblock,
38:22it's nothing different to biopsies.
38:25But when we talk about smears,
38:27be aware of the three dimensionality
38:29of psychological smears.
38:30So in this case this is a nice
38:32staining of the membrane of the cells,
38:34but here the the cells are more crowded,
38:37so we can have a false impression
38:39of cytoplasmic staining.
38:41We see which is not real,
38:42it's because the cells are overlap.
38:46And and in the in the in the report
38:50is it recommended to specify the type
38:52of psychological sample cell blocks
38:54smear wherever the clone use and the
38:57controlled use at least we have to
39:00evaluate 10 evaluable tumor cells
39:02no sorry 100 cells not 100 cell,
39:06100 evaluable tumor cells and very
39:09important again pre analytics and
39:11we have to use appropriate controls
39:14of course for biopsies we use.
39:16Tonsil, placenta, whatever.
39:18But for a cell blocks we decide
39:21to use placenta.
39:22So we take a placenta every
39:24month and we make small pieces to
39:27do cellblock and we make many,
39:29many many imprints and we
39:32stand with papanikolau.
39:34So we use smear or smear
39:37and cellblock or cellblock.
39:40OK.
39:41And these are some examples on
39:43nice examples of period while
39:45on smears the macrophages.
39:47But for example,
39:48what happened with this case?
39:49Things are not easy.
39:50This group is positive,
39:52but what this weather ourselves?
39:53Macrophages probably,
39:55but stain membrane and cytoplasm
39:59pants everything but it be be aware
40:03that some normal bronchial cells
40:05can also be stained with PDL one.
40:07OK, so it's a.
40:09What is my recommendation when we use
40:12PDL one on psychological samples?
40:15Caution.
40:15Be cautious because it's an important issue.
40:19OK, we have to waste a lot of time
40:23interpreting video one and psychological.
40:26What happened in Europe regarding
40:29immunohistochemistry in general?
40:30This is a paper Paris 2020 about a survey.
40:37Our Community subcommittee,
40:38how you measure is performed in
40:40different laboratories across Europe.
40:42So they said that mother clinical
40:45practice requires immunohistochemistry
40:47more and more and psychological samples.
40:50And to be to be able to do
40:53immunohistochemistry in accurately way,
40:55laboratories have to be able
40:57to optimize and standardize the
40:59preparation of psychological samples.
41:02They have to optimize and validate
41:04immunohistochemistry staining protocols
41:05and they have to use appropriate.
41:07The controls and of course they have to
41:10follow external quality control problems.
41:12This is very,
41:14very difficult as you say.
41:16Is
41:1922145 to 45 laps participate in this survey?
41:25And look at the differences in fixatives.
41:27Cell block is fixed mainly informally
41:30but there are other fixed actors,
41:32cytospin, smears and liquid based.
41:35There is a very variation in fixation.
41:38It's unbelievable but it is true.
41:41This is a lot of variation in in
41:44the staining the staining before
41:46perform immunohistochemistry.
41:48Some are in liquid base,
41:51cytospin and smear.
41:52There is variation in multiple
41:54samples and also there is.
41:56Variation and how the samples are
41:58storaged before doing amnestic chemistry.
42:01So if that that happened in Europe,
42:03I cannot think what happened
42:05in the in the entire world.
42:08But you know cell blocks are are the first
42:11option for us as a cytopathologist to do
42:14all kind of immune or whatever right.
42:17But cell blocks also have some
42:19problems because there is a regarding
42:21PDL one there is atherogenic city
42:24interpretations of course in their
42:26crunch we know that inter pathologies.
42:28So this is a reality and it's
42:31a reality in in every way.
42:33So can can what can we do
42:36to overcome this challenge.
42:39What we can do?
42:40Two things based on literature,
42:42but before that I want to.
42:44Go or let you go to the South
42:47of Spain this is Cordova.
42:50In the very South of Spain this is Neta
42:52de Cordova and this called patios.
42:54Patios plenty of flowers that are
42:57very famous and are very beautiful.
42:59And this is Granada appearing in Granada.
43:02I don't know this is the Alhambra of
43:04Granada is a wonderful place to visit,
43:06so I'll invite all of you to them.
43:09First solution,
43:10first solution came from a paper
43:13recently published is published is here.
43:15Up in 2022, this year and they say.
43:20Is true the impact of of a pathologist
43:22personality on the interior
43:24server variability and diagnosis,
43:26accuracy of predictivity.
43:27We're one immunochemistry in Lancaster,
43:29OK, it's very recent paper.
43:32The authors make an hypothesis saying
43:37that pathologists personality has a lot
43:40to do in this interservice variability
43:42and then in the diagnosis accuracy
43:45of PDL 1 immunity immunity scoring.
43:47So they they they the paper is done in
43:50this way 17 pathologist for four people,
43:53one in my scoring in 50 resected
43:55resected a resection non small cell lung
43:58cancer and this pathology is completed
44:00that certified personality test.
44:03That assess.
44:04Different type of personality,
44:06neuroticism, extraversion,
44:08etcetera are here.
44:10So good concordance was obtained
44:13in cases entirely positive,
44:16so less than 1% or very high positive,
44:20negative or positive.
44:21But the problem was in the
44:24middle for cases with PDR one
44:26score around the cutoff value.
44:28The conclusion of this paper look at
44:31this is published in lung cancer in 2022.
44:34Pathologists with higher score
44:37get higher score pathologies with
44:40a personality careful diligent
44:42conscientious anness pathologies.
44:44By contrast a lower score a big problems
44:48came with a pathologist that have.
44:52Notice, notice, personality.
44:54I'm not inventing anything.
44:57This is published.
45:00And she was sensitive or whatever.
45:02So I don't know what type
45:04of personality you have,
45:05but. I I personally won't go through this
45:10certified personality test just in case
45:13the second solution I think more more.
45:18Real, I would say is to have help of the
45:21digital pathology because some digital
45:24pathology system have an algorithm for
45:27helping us in the interpretation of PDL one.
45:31We use this UDC 200 system from roads
45:36that has an algorithm to evaluate
45:39PDL one with the clone speed 263.
45:44A digital purchase pathology is
45:46very important also in psychology
45:48you have many many advantages.
45:50I would say we can use it as a
45:52telepathology consultation, discussion,
45:55discussion of cases with some other expert.
45:57We can do as a teaching tool.
46:01And as you see before we can immortalize
46:04the smear for do other ancillary
46:06testing and we can use algorithms,
46:08but but to use algorithms we don't.
46:13We can use a smear.
46:14We is mandatory to use cell blocks,
46:17paraffin cell blocks.
46:18So it is necessary to get adequate
46:20cell cell blocks and if possible to
46:23preserve the architecture of the sample.
46:25So why is important the management
46:27of the samples and the development
46:29of these new needles?
46:31And these new procedures,
46:32OK so this is a small study we
46:36perform in our laboratory using
46:38evaluating 32 cell blocks by eye in
46:42the microscope and scan these cases
46:44and and go through the algorithm from
46:48our system and see what happened.
46:52This is an example.
46:53You see the example of the cell block.
46:56We select this area,
46:58we teach the machine how to,
47:01how to interpret,
47:02how to select the cells.
47:04So we teach the machine and
47:07the machine teach teach us to.
47:09So in this particular area
47:12region of interest,
47:13the percentage of PDL one is here 39.8.
47:17We can select many different areas,
47:19we select another one
47:21and here the percentage.
47:22This is a I can see he's 34
47:28something 3033.3 so at the end.
47:31We can do with many many other
47:33areas of the sample.
47:34The the machine gives us a
47:36like a medium results.
47:38In this case the machine says that the
47:41person is of positivity of 137.6 OK.
47:45So at the end in this study,
47:47what we did is.
47:50Demonstrate is a very short number
47:53of cases that the concordance
47:55among pathologists sticks to.
47:57X1X2 and X3 are by ice.
48:00A artificial intelligence one and AI two are.
48:05It is the same samples analyzed
48:08using this algorithm,
48:10the Concordia single,
48:11but is better when you use this
48:14algorithm and of course is for
48:16almost perfect when the algorithm
48:18is compared with the algorithm.
48:21What we did is to review blinded 3
48:24months later same cases, so pathology 1,
48:27pathology 2 using the eyes,
48:29artificial intelligent pathology to using
48:33the eye and artificial intelligence.
48:36Thing changed a little because
48:38it seems that using artificial
48:40intelligence you can be more precise.
48:43Be careful because we have to teach
48:45the machine before doing all this.
48:48The machine doesn't know if
48:51we don't teach the machine.
48:52So remember human intelligence
48:53has to be at my heart
48:55with artificial interest.
48:57So just finishing this part
48:59of immunity second mystery.
49:00Sometimes we try to do our best,
49:03but sometimes sometimes it
49:04doesn't work because the.
49:06Sample because of whatever.
49:08Be be aware that CPS and PDL
49:11one is not possible to measure
49:15on psychological samples.
49:16We only can evaluate 2 more cells,
49:20not infiltrate, it's not in.
49:25Lymphocytes and so on.
49:27And sometimes we do as best as we can.
49:31But simply immunity,
49:32the mystery doesn't work.
49:33That happened, but with adequate samples,
49:36proper management of the samples and
49:39appropriate validation controls,
49:40it really works on smears.
49:42And you have here some examples
49:45from real cases from our department
49:48and cellblocks and smears.
49:51So yes, Sir,
49:522 words about this piece is very.
49:55Useful on psychology because
49:57especially on smears,
49:59because the main advantage is is
50:01that we have the entire nuclei,
50:03so the signals we analyze at the real ones.
50:08What we do is to select this monolayer
50:11areas and avoid these crowded because
50:13here we cannot interpret any signal.
50:16Here yes we we have the isolated
50:19nuclei to interpret the signals.
50:21What we do is to to to
50:24mark this the the smear.
50:26So it's not cost effective to to
50:28put the probe over the whole smear.
50:31So we mark the areas with the cells are
50:34monolayers even we can do two different.
50:37Approves and one on this part
50:39of this meal and one of the
50:42other part of this smears.
50:44Probably the most tricky issue
50:46on smears during fees is they is
50:49optimizing the Pepsi in time because
50:51if there is insufficient digestion
50:54the proof doesn't enter into nuclei
50:57and we see nothing but the the
51:00the digestion is is too strong
51:02is the sample is over digested.
51:05We have this like a Donuts.
51:08Appearance of this is and this is not,
51:11we cannot see nothing.
51:13There are many papers published
51:15in the literature that that show
51:19the the usefulness the of this.
51:23It's me.
51:24Our support to infuse this is an
51:25example of ALK is that we do this
51:27is the real image we see at the
51:30Microsoft that fluorescent microscope
51:31and the very fancy, you know,
51:33makeup image that is very nice.
51:35But we deal every day with this
51:38type of images.
51:40This is a rogue one positive cases and
51:43this is a polysemic negative for Ark.
51:46Sometimes we we see not entirely negative
51:50cases and in which the chemistry for all.
51:54We found out these polysemic cases and fish.
51:57So as I said before, the advantage is
52:00that we we have the entire nuclei,
52:03we have to test at least 50 cells,
52:06is better to test 100,
52:07we test always 100 cells.
52:09Of course digital scan the preparation
52:13because we can understand.
52:16And yes,
52:16I have some words about NGS,
52:18but before going to NGS we are
52:21going to Canary Island.
52:22The Spain have two different island.
52:25This is Canary Island without in the
52:27South close to Africa in the in the
52:30Atlantic Ocean and this is island
52:31of Tenerife is a totally volcanic
52:34island as you see here and we have.
52:37Beautiful places,
52:38so you are also invited to
52:40go to Canary Island.
52:42So what about NDS and and
52:45psychology was the same.
52:47There is a significant variation
52:48in the quality of the tissue,
52:50diversity in specimen preparation,
52:53and lack of standardization
52:55across laboratories.
52:56It's important to say that if it is feasible,
53:01obtaining concurring coronaviruses and
53:03effeminate to maximize chances of sufficient
53:05material to perform ancillary studies.
53:07Please recommend it.
53:10We have to to to take into account when
53:14we talk about NDS to many to two ideas.
53:18One is overall cellularity that translate
53:21total DNA gene so it belongs to all
53:24nucleated cells in the samples and
53:27the other term is tumor fraction that
53:30translates to analytical sensitivity
53:32that means the percentage of tumor
53:35cells on the the whole proportion
53:37of cell on the on the summers so.
53:40How many cells do we need to get success to
53:44get successful with NGS is easy to calculate.
53:471 cells have about 6 to 76 to 7.
53:53Sorry. Picograms of DNA,
53:55molecular acid required in one
53:58nanogram of DNA a input needs.
54:02One about 1:50 in the cells.
54:05So for example NGS ion torrent that
54:09requires around 10 nanograms of DNA
54:11for the panel of 53 genes therefore
54:15will require approximately 100.
54:18Sorry as 1050 hundred 500 sales more
54:23or less following this calculation.
54:25OK, we are talking about sells all
54:28kind of cells but the point is.
54:31To detect mutation in samples that
54:34have tumor cells and normal cells.
54:37So we have to enrich the samples and
54:39there are different techniques to
54:42enrich sample manual microdissection,
54:44manual microdissection and laser
54:46capture at this section.
54:49So we can get good results during NGS
54:51doing even digestion on the on the
54:54smears of of course the cell block.
54:56So The thing is that the the points
54:59are appropriate to more fraction.
55:01This is the most the key.
55:02The key issue to get to determine
55:06the successful mutation test.
55:08So for tests that required between
55:115:00 and 10:00 nanograms of DNA,
55:14RNA is published in the literature
55:16that psychology is the sample
55:18is a psychological samples.
55:20We need about 200 tumor cells,
55:23not all kind of cell tumor cells.
55:26On with a 10% of two more cell content,
55:29if it's paraffin we we would
55:33need 502 more cells,
55:35which means that about
55:372020% of tumor cell contact.
55:39This is published in the literature
55:41and this is already validated.
55:43It's validated by the international
55:45Molecular Cytopathology Meeting Group
55:48which is led by Professor Giancarlo
55:50Trongone from Naples in Italy.
55:52And we many institutions around the
55:55world are participating in this.
55:57Are these in these validation aside
55:59we published already to to studies
56:02validating mutations and last year
56:05we published the study validating
56:07translocation so and and this
56:09validated on smears only smears.
56:12OK so to finish this part and yes
56:14it's useful tool for the analysis
56:17and molecular alteration and
56:19symbological samples.
56:20It allows for a simultaneous
56:22analysis of several genes the quality
56:25of DNA and everyone is.
56:27Better because it's not formally
56:30in our paraffin went through and
56:33we need of course externalization
56:36protocols and internal validation.
56:39So it is important is to cheat or
56:42to manage a small sample using
56:44different specimens, biopsies,
56:46core biopsies and smear.
56:49Anything is good because if we
56:51have for example this cell block,
56:53it is good, it's very cellular,
56:55we can do whatever immuno fees are.
56:57And yes, but what happening
56:59if we have this cell block,
57:01probably the two more content is
57:04very low for performing an NGS.
57:06But if we have smears from this
57:08patient we can scan and we can add.
57:11So putting together all the
57:15samples we will be successful.
57:18Doing anything immuno in the cell block fees
57:22or NGS on the on the smears in this case.
57:26So the success of any test
57:29depends of the tissue procurement.
57:33The test that we selected
57:35related to amount of tissue and
57:37characteristic of this the tissue,
57:39the tissue of the samples and a careful
57:44interpretation of the results because
57:46it the treatment depends on that.
57:49So, but this is very nice and very useful.
57:54Very.
57:54But so far,
57:55this is the real world and we live there is.
58:00Psychological samples are under
58:02use for molecular tests because,
58:04importantly, there is a lack of
58:07standardization across different labs.
58:09For specimen collection and processing,
58:11there is kind of reluctance of
58:14molecular and immune labs to validate
58:17different types of cytological samples.
58:19This is a kind of lack of awareness
58:22among oncology and pathology communities
58:24regarding the utility of this type
58:27of samples for biomarker testing.
58:29And even there is reluctance of some
58:33pathologists and cytopathologist to
58:34sacrifice these smears and this is
58:37true although we have some solutions.
58:39So yes, to finish and talk, yes, 2 minutes.
58:43About macro environment,
58:44I don't know you have had some time
58:46and I like to go up to this pain
58:49because this is the region where
58:51where I was born is close to France,
58:54is in the Pyrenees.
58:55And it's beautiful in winter
58:58and it's beautiful in summer,
59:00so you also are invited to
59:03the north of Spain.
59:05And just few words because I
59:07think it's very important tumor
59:10microenvironment because cancer is
59:12made not only of tumor cells that it
59:15is with a tons of characteristics,
59:17but this also made these cells are in
59:20a in a background in an environment
59:23that has blood lymphatic cells,
59:25macrophages, B cells,
59:26T cell dendritic etcetera,
59:28etcetera.
59:29So the oncology is changing because
59:32the historical concept of of.
59:35Developing drugs or was the drug
59:37has to target tumor cells.
59:39But now the new concept is the drug
59:41target the immune cells and the immune
59:44cells kills the the immune cells,
59:46kills the the tumor cells.
59:48So if we are able to understand the
59:52new context of malignant tumors this
59:55is important for design effective
59:58anti cancer immunotherapies.
01:00:02We know that there are tumors
01:00:04that are are not inflamed,
01:00:06they don't have many inflammatory cells
01:00:08related with the tumor cells and these
01:00:11tumor do not respond to immunotherapy.
01:00:14By contrast that they had
01:00:15hot tumors that have a lot of
01:00:18tumor infiltrating lymphocyte.
01:00:20In general they express PDL one and
01:00:22more than the others they have a
01:00:26more genomic instability etcetera.
01:00:28These two more to response
01:00:30to immunotherapy they key.
01:00:32Issue is how to do transport
01:00:35this tumor into other like this,
01:00:37we don't know so far.
01:00:39So we have to deal with with what we have.
01:00:43So I said before PDL one, win 4,
01:00:46PDL one we've seen only in
01:00:49ministry chemistry but is has
01:00:50many limitation as we know.
01:00:52So the assessment of tumor infiltrating
01:00:55lymphocytes together with PDL 1 by
01:00:58Minister chemistry could improve
01:01:00the specificity of this essay.
01:01:02How do you test this micro environment
01:01:05in the real day in pathology using
01:01:08this technique that is called
01:01:10Multiplex immunofluorescence,
01:01:11immunofluorescence,
01:01:12fluorescence that the technique
01:01:15is very simple.
01:01:17It's not simple but is to do several
01:01:20runs of immunofluorescence each one
01:01:22mark with a specific mark with a
01:01:25different color SO1 above the other.
01:01:28We can do 68.
01:01:30Up to in our department up to 8 up to 10.
01:01:34So the end of the story is this
01:01:36we it can have the the separate
01:01:39antibodies mark here but we can the
01:01:42machine can merge all these images
01:01:45so we can see the different cells
01:01:48population of the cells in the in the.
01:01:51In the stroma of the tumor and
01:01:53even the relationship between
01:01:55these cells and the tumor cells,
01:01:57and we can quantify also these cells,
01:02:00but the question is it is feasible
01:02:03and psychological samples.
01:02:05We need formalin fixed paraffin
01:02:07embedded tissue and we need
01:02:09amatoxin analysin as a guide.
01:02:11We the architecture of the lesion
01:02:13is required and also the present of
01:02:16the stroma because in the stroma is
01:02:19mainly where the inflammatory cells are so.
01:02:22Go back to the the first part of my
01:02:25talk the the need to get good if it
01:02:28is possible diet cell blocks using
01:02:30this new needles and new procedures.
01:02:33This is very very important
01:02:35actually with this.
01:02:36These are real examples the
01:02:38amatoxin and the and the study when
01:02:41we use all these antibodies.
01:02:43And we published this study last
01:02:46year and is even is not a validation
01:02:50is nothing easy as 5-6 cases.
01:02:52I think yes to show that there
01:02:55is possible using this technique
01:02:57is is feasible using this type of
01:03:00psychological samples and even we can
01:03:03measure we can count the percentage of
01:03:06different immune cells in each case.
01:03:09This is 1 case with many cells CD20,
01:03:12CD, a CD3 and.
01:03:13This is another case with
01:03:15low low number of cells.
01:03:16Same with CD88 and CD11B.
01:03:22And this is the the the paper and the
01:03:24the paper that published recently with
01:03:26Fernando Smith that we refer also to
01:03:29this to this technique and this is
01:03:31one of the picture of the paper you
01:03:34see here and these cells are here.
01:03:37So yes my last my last slide just
01:03:39to summarize I would say there is a
01:03:43theological samples provide versatile
01:03:45ways to perform properly accurate
01:03:47diagnosis as well as biomarker
01:03:49testing for lung lung cancer.
01:03:52Analytical case,
01:03:53you as in the specimen collection and
01:03:55and and management of the samples
01:03:58impact the success of diagnostic
01:04:00biomarker testing.
01:04:01So the cyto technician have a
01:04:04key role on all this process.
01:04:06We are nothing without them and they
01:04:09pathologists and the psychopathologies
01:04:11of course plays a key role ensuring
01:04:14the success of biomarker testing.
01:04:16So we need to be able to integrate clinical
01:04:20radiologic morphological biomarker results.
01:04:23And just to remind you that precision
01:04:25medicine is in the hands of the,
01:04:27in this case, cytopathologist.
01:04:29So this I want to thank
01:04:31you for your attention,
01:04:33for your patience with my English.
01:04:35And this is my university,
01:04:37University of Navarra is a beautiful place.
01:04:39Of course, you all are invited seriously,
01:04:42you are invited to my university.
01:04:44So thank you very much.
01:04:45I'm hoping for your question.
01:04:54Any question? No.
01:05:01The meeting. You need to have
01:05:06the clinical folks through that.
01:05:10You know, for their rose
01:05:11technique. In other words,
01:05:13how did you implement that new
01:05:14needle and how teams embrace it?
01:05:17Yeah, it would in our in our university
01:05:21they then what we did is that the ECHO
01:05:24and the Echo endoscopist start doing
01:05:27that because it's easy doing doing use
01:05:31and then the ebus people follow them.
01:05:35So yes, they start using it and we
01:05:39were getting very good results.
01:05:41So we implement so we use
01:05:44in every procedure now.
01:05:46And they were very amenable to change.
01:05:49Nothing, no, no, no, no changes,
01:05:51no change. No, no, no.
01:05:53It's not a big issue,
01:05:54the cars, but the only,
01:05:56the only thing is that the person who
01:05:58do that has to be trained and has to do,
01:06:01you know, but it's very good.
01:06:06I don't know. There are several,
01:06:08several companies doing that. Yeah,
01:06:10I I I can give you this data by e-mail.
01:06:19You know one species, but all the clinical
01:06:22trials were done on tissue specimens.
01:06:25How do you know that the psychologist has
01:06:27the same association? No. Well.
01:06:33Yeah, so people one is even is not
01:06:36validated in psychology, but even is not.
01:06:40Consider in psychology, I would say.
01:06:43But I know yeah, should be.
01:06:45Of course you'd be under. Yes.
01:06:49No, no, no, no. No, no, no, no.
01:06:53Yeah, it's it's a proper tissue,
01:06:54but it's going to be a proper for cytology,
01:06:56I hope. Yeah, but no, yeah,
01:07:00there are papers in the
01:07:02literature that demonstrate the,
01:07:04the good results comparing resection,
01:07:06specimen, cellblock and smears that
01:07:09exist many papers one and the second
01:07:13thing is that there are coming papers,
01:07:17we are doing a study.
01:07:18I will let you know when we finish.
01:07:21Will be the one on cytological samples by I,
01:07:25by the algorithm that I show you
01:07:27and compare with clinical results.
01:07:30This is the study we are doing.
01:07:32So yes, I'll let you know and we don't so,
01:07:36but yeah, yeah,
01:07:37it's not validated by I think the
01:07:39patient would benefit if it's well done.
01:07:44Yeah. No, no, no, no. Not yet.
01:07:45Not yet. Yeah, you are right. Not yet.
01:07:48Hope we will have, but not yet.
01:07:52Multiplex.
01:07:56Help.
01:07:58Because now I think.
01:08:00Yeah, it's more research in our
01:08:02in our lab is only researched,
01:08:05no translation to the clinics. Against the.
01:08:14Yeah, so far is same with PDL one,
01:08:17there is no translation to but even
01:08:19in in surgical specimen there is no
01:08:22translation so far with Multiplex, yeah.
01:08:29I just requested since now
01:08:32they finally go by option.
01:08:36You know, now we get assessment report.
01:08:41What's the best way
01:08:42to process those tests
01:08:44because they all institute?
01:08:47You know, sometimes, you know.
01:08:51Formatting. Start with that.
01:08:57At all.
01:09:00Awesome. Thanks. So
01:09:03that you know what's that?
01:09:08Well, the the first thing is that all
01:09:11sell block is that recommendation
01:09:14should be fixed in informally.
01:09:17The small biopsies and cell block
01:09:19that we clot whatever we do,
01:09:21the cell block should be facing formally
01:09:24because it allows us to adapt to
01:09:27the protocols of immune or whatever.
01:09:30So first thing has they should
01:09:32be fixed in formalin.
01:09:33Yeah it's the main recommendation, yeah.
01:09:36And then yes trade like a paraffin,
01:09:40normal paraffin sample.
01:09:42So
01:09:42that little spectrum will state.
01:09:46Well, it depends of the hospital.
01:09:48In my hospital is done by the
01:09:51we are our small group, so we,
01:09:53we psychologists do both the
01:09:56small biopsies and the psychology.
01:09:59We consult with other colleagues
01:10:01that we are handled by us. Yeah.
01:10:03In some other hospital is you know,
01:10:06the problem with psychopathology is
01:10:08that there is it's very hard to get
01:10:10standardized procedures, it's very hard so.
01:10:14Yeah, we have to do.
01:10:15We have to work a lot. Yeah.
01:10:22OK. Anything.