Pathology Grand Rounds: November 10, 2022
November 15, 2022Information
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.