“Once in a Blue…Book” A case-based discussion of rare disorders in Hematology

[Music] [Music] so [Music] [Music] so [Music] [Music] foreign [Music] [Music] [Music] hmm [Music] [Music] [Music] [Music] [Music] [Music] do [Music] so [Music] [Music] so [Music] [Music] [Music] [Music] so [Music] so [Music] [Music] so [Music] man [Music] [Music] [Music] do [Music] [Music] [Music] so [Music] [Music] do [Music] so [Music] [Music] foreign [Music] [Music] [Music] so [Music] [Music] [Music] foreign [Music] [Music] [Music] so [Music] [Music] [Music] [Music] foreign [Music] [Music] [Music] [Music] [Music] so [Music] so [Music] [Music] do [Music] [Music] so [Music] [Music] me [Music] [Music] so [Music] [Music] so [Music] good morning colleagues and friends. This webinar is brought to you by Mumbai Hematology Group. It is supported by Intas and managed by Mice Ideas. I thank Mr. Dr. Manoj Kumar and his team from Intas, Mr. Rajesh Sharma, Kalpash Pamba and team from Mice Ideas. Executive Committee of Mumbai Hematology Group, our Chief Guest today, Dr. Tejinder Singh from Delhi. Our guest speakers today, Dr. Swati Pai, Dr. Roshini Shekhar, and Dr. Vishwa K.S. from Manipal Hospital, Bangalore. All our discussions were eminent hematologists and hematopathologists. New participants for sparing your Sunday morning. From tomorrow, I am sure you remember that we have Best of ASH, which is one week long webinar every day from Monday to Sunday, beginning somewhere in the evening hours at variable times: 6:15, 5:30, 6:15, 6:15, 5:45, 5:30 from Monday to Saturday, and on Sundays, the whole day from 10:15 to 6:30. You already have the agenda with you in your email box. Guest speakers, it's my privilege to introduce them to you. You all know them. Dr. Swati Pai is Consultant Hematopathologist, Department of Lab Medicine, Manipal Hospital, Bangalore. She is going to lecture today on Systemic Mastocytosis. Dr. Roshini Shekhar from the same department, she's Consultant Hematopathologist. She's going to lecture on Transient Abnormal Myelopoiesis. Dr. Vishwa K.S., once again, Consultant Hematopathologist from the same department, is going to speak on VPDCM. Our discussants today are from Mumbai, Delhi. Dr. Vidisha Mahajan from the same institute, Mumbai.

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Dr. Sanjeev Gupta from AIIMS, New Delhi. Dr. Narendra Tejwani from Rajiv Gandhi Cancer Institute, Delhi. Dr. Shah Sagar from AIIMS, Delhi. Dr. Amrita Saraf from Sir Ganga Ram Hospital, Delhi. Dr. Rama Hariharan from AIIMS, Delhi. From ESI-PGIMSR, Maniktala, Kolkata.

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Vedanta, Ahmedabad. Our Sunday quiz for everyone. As usual, there will be nine statements. Today, it is regarding Rosai-Dorfman Destombes disease, RDD disease. One of the statements will be wrong. You have to pick up the number of that statement and email it to mb Agarwal1@gmail.com. Winner will be fastest finger first. So, here we go with the nine statements. On the top is the email ID to which you will email one of these numbers, which statement is a wrong statement. So, Rosai-Dorfman was first described by Destombes, a French pathologist, 1965. Two, it is also known as sinus histiocytosis with massive lymphadenopathy. Three, the mean age of presentation is 60 years. Four, patients usually present with bilateral massive painless cervical lymphadenopathy. Five, almost 40 percent of patients present with extranodal disease. Six, Rosai-Dorfman is often associated with autoimmune, hereditary, and or malignant diseases. Seven, histiocytes are S100+, facing+, CD68+, CD1a negative, and 2007 negative. Eight, the cells often show emperipolesis, that is, trafficking of intact leukocytes through their cytoplasm. And nine, the treatment includes observation alone, or surgical resection, or steroids, or immunomodulatory drugs, or chemotherapy, or radiotherapy. I'll keep this slide on for a few seconds. One of these statements is wrong. Once you find out that statement, the number of that, you will be emailing to md Agarwal@gmail.com. Fastest finger first is the winner. Okay, that's the email ID. The next job this morning is to present the case of the week. So, this is a very interesting case, and we are very happy that we could help this gentleman. 21-year-old male from Pune, admitted for a prolonged illness of two months with almost daily fever in these two months. Lost 12 kg weight and had extreme fatigue. The family noticed icterus for a week. Originally, he was a resident of Tripura and he had come to Pune about 18 months ago for his education. He had no it habits. A bit pale. He was more active. There were no lymph nodes. There was no liver, no spleen. Pancytopenia: 8.7, 2,796,000 is frank hyperbilirubinemia, direct more than indirect. AST-75, ALT-180, very high alkaline phosphatase, low albumin, normal globulin. CT showed mild hepatomegaly with hepatic steatosis and no dilatation of the biliary tract. Workup for fever done in Pune and in Bombay, negative. He had received plenty of broad-spectrum antibiotics and antifungal agents in Pune. Before coming to Mumbai, he had undergone a bone marrow aspirate in Pune, which was non-contributory. The workup for all sorts of hepatitis viruses from A to E, CMV IgM, EB virus IgM, COVID, all negative. Workup for autoimmune hepatitis, negative. A liver biopsy was done, which showed cholestasis. There was no hepatitis and no cirrhosis. Patient developed oliguric renal failure, and he needed adrenal replacement therapy on multiple occasions. Pancytopenia worsened. He developed coagulopathy, which suggested consumptive coagulopathy. On day 9, we received the EB virus PCR report, and it was positive with more than a million copies. By now, peripheral smear, besides pancytopenia, started showing mild neoplastic reaction. His ferritin went to beyond 5000. LDH was high. Triglyceride was high. Fibrinogen was low. The marrow was repeated at this stage. It was dry. With the data mentioned just now, HLH was suspected, almost diagnosed, and he was given steroids in the form of dexamethasone. At present, HLH was secondary to an undiagnosed entity. After starting dexamethasone, he had a remarkable response, and within half a day, he became afebrile for the first time in his two months illness, and his vitals stabilized very quickly. On day 13, his bone marrow refined biopsy report was received. It confirmed HLH. In addition, there were large areas of necrosis. The interstitial keyboard positive lymphoid infiltrate composed predominantly of malignant looking cells with an IHC showed changes suggestive of NK cells: CD56+, 2+, 7+, partial CD3 negative, 4 negative, 5 negative, 8 negative, 16 negative, 57 negative, TCR negative. A diagnosis of LGL leukemia was excluded because CD3 was negative, CD8 was negative, 56 was negative. Liver biopsy was again looked at, and once you knew what you were looking for, one found there is sinusoidal EB virus positive NK cell infiltration there as well, and in fact, there was also occasional hemophagocytosis, and these were totally missed earlier. So, diagnosis of aggressive NK cell leukemia, which secondary HLH was made. Patient was treated with dexamethasone, which he was already on. He was given IV gamma globulin. His condition stabilized, and then he received etoposide. He further stabilized, and then he received treatment for his NKCL with first just monotherapy, PEG-asparaginase for two doses, and subsequently he received attenuated SMILE: SMILE is dexamethasone, methotrexate, ifosfamide, asparaginase, etoposide, no adriamycin. He got two cycles of about 60 percent of the dose, and this was followed by nivolumab, six doses at three weekly intervals. He had stabilized with SMILE again alone, and these nivolumab were given mainly while waiting for the allo transplant. He was lucky enough to have a matched sibling donor. He underwent an allo transplant at another center successfully. Has gone back to now Tripura for rehabilitation and is under supervision of the local medical oncologist. A rare disease with a common complication and with a rare outcome, that is, survival. So, that is our case for the day. This is HLH, erythrophagocytosis. That is his bone marrow biopsy, low power view. That's high power view showing interstitial neoplastic infiltration. Cells show moderate cytoplasm, irregular nuclear contour, and prominent nuclei. That's CD202 positivity, and that's EBER positivity. This is a flow diagram for the oncologists or hepato-oncologists for treatment. NKCL, younger patient, lymphocyte count more than 500, no organ dysfunction, can go on SMILE. When you receive CR, you can go for auto. If you don't achieve CR, allo or a clinical trial. If you are more than 70 or if the lymphocyte count is low or there is organ dysfunction, you can take your decision of treating with reduced dose SMILE or even sometimes L-asparaginase alone or any other asparaginase containing regimen. If you don't achieve CR, you can go to a salvage chemotherapy to auto to allo or just a palliative therapy depending upon your age etc. The best supported therapy, most likely the cure is possible only if you use an asparaginase based regimen followed by an allo transplant. Nivolumab is a new addition to the treatment because these cells invariably express PDL. My most important part this morning is to invite our Chief Guest to inaugurate our webinar, and we are blessed by the presence of a very, very senior personality and a very important personality and a very famous personality in the form of Dr. Tejinder Singh, a guru of all hematopathologists in this country. Who does not know him? But I have to just complete the formalities of introducing a real guru of hematopathologists in this country. I've had occasion of listening to him, especially on the bone marrow to find biopsies. It's a treat to listen to his competence, his power of teaching, and that is much more important than what is listed on this slide. Anyway, he is the present Senior Consultant, Quest Lab, Delhi. Formerly, he was Dean, Faculty of Medical Sciences, Delhi University. Formerly, he was Professor and Head, Department of Pathology at MMC, Delhi. Past President of Indian Association of Pathologists and Microbiologists. Also Past President of ISHBT. Fellow of Indian College of Pathologists. Fellow of Indian Society of Hematology and Blood Transfusion. And his author of seven books in hematology. At the pleasure of being his co-examiner in AIIMS, and I think that's the longest period I spent with him, and what wonderful it was to interact with him, not only on medical issues but also on various non-medical social issues. Well, thank you so much, Dr. Tejinder Singh, for accepting this invitation and blessing us to inaugurate our webinar and give some words of wisdom to our students and others. Over to you, Professor.

Good morning, everybody. And thank you so much, Professor Agarwal, for giving me this honor. And I'm happy and pleased to inaugurate this hematopathology session. As we all know that all hematologists across this country, they appreciate the initiative taken by you and then sustained for so many months at a stretch by hematology teaching. And this hematology teaching is by renowned hematologists across the globe. As of now, our clinical hematology is at par with the best in the world, and the latest drugs are in use. The role of a hematopathologist in correct diagnosis and accurate assessment of response to therapy is very, very crucial, and more so in hematologic malignancies where the response to therapy has to be seen repeatedly. In an ideal scenario, the expensive genetic and molecular tests need to be carried out keeping the morphology in mind. See, for example, PML-RARα in APML, inversion 16 in M4Eo, or Annexin A1 in suspected Hairy Cell Leukemia, NPM1, FLT3 whenever we have AML with the big cup like inclusions, or JAK2 in polycythemia. Better if we do that, that avoids unnecessary testing and extra financial burden for the family. But now, the diagnosis is not based only on morphology, but it is based on integration of morphology, immunohistochemistry, flow cytometry, cytogenetics, FISH, and even NGS. Therefore, we need pathologists who are trained in various subspecialties and also to integrate the clinical morphology and the molecular for exact diagnosis of a particular case. Now, do we have such hematopathologists? Yes, we do have, but a handful of them. You'll be surprised to know that there are only 10 seats in DM Hematopathology in this country. However, for clinical hematology and clinical hematology, we have about 50 seats and a few fellowships across this country. To keep pace with the clinical hematology, we need to impart training to our pathologists colleagues in various subspecialties like flow, molecular, and other modalities. So, what we should do is we should start DM Hematopathology and also fellowships in periods varying from three months to one year. Now, that is where the role of experienced teachers and institutions like AIIMS, PGI, CMC, and Tata Centers comes into play. They should set up, and not only that, there are very good private hospitals who have got very nice hematology setups. They should start the fellowship program varying from three months to six months, maybe one year. Now, that will improve, I'm very, very sure that this approach will go a long way in improving not only the diagnostics but also the management of hematologic disorders. Thank you very much. Thank you, Dr. Tejinder Singh, for those golden words, very inspiring, and I'm sure each word of that has been heard by the PGs, trainees, and fellows, as well as the seniors, and they will imbibe it and act on it. So, this message was fantastic, and once again, I'm grateful to you for blessing us these three minutes, and this brief speech was very, very important to us. Now, we go to the medical session of the day, and our first speaker is Dr. Swati Pai. She's going to speak to us on Systemic Mastocytosis.

Thank you, sir. Thank you for inviting me, uh, along with my team. This is one of the unique sessions where all three of us who are reporting in Manipal Hospital, Department of Hematopathology, are speaking today. I'm honored to be speaking under your, as well as having Dr. Tejinder, one of the, you know, the prime people who taught us morphology. And as you see our cases, morphology will remain the gold standard for, uh, diagnosis. I will start my session. Okay. So, um, I'm sharing my screen. I think it's visible, right? Yes. Yeah. So, we came up with this idea of doing this session, and sir, you know, reached out to us, and actually, we thought that let's address the problem areas in diagnosis. And my request to the discussants is, please share. I have a knowledge sharing session because these are not lesions where if somebody asks me, in your experience, because I have an experience of one or two in such cases. So, we came up with this once in a blue moon, kind of cases which we rarely see, but when we see them, as you will see in our cases, it takes so long to make a diagnosis, and these are issues which have serious, uh, clinical implications on the patient. So, I'm going to start with two. I will present two cases. Please remember that, you know, that, uh, I'm talking about systemic mastocytosis, but when I saw this case, I didn't know that I am dealing with systemic mastocytosis. So, our first case is a 23-year-old female who came to us from Mauritius. She was 23 when she came to us. She had ALL when she was three years old. She was fully treated in Mauritius, but in March 2013, she presented with a low back ache and pancytopenia. A bone marrow which was done there wasn't conclusive. For whatever reason, they decided to treat her as a relapse case, and they treated her with 6MP and methotrexate. She didn't really respond. She had a massive spleen, and they did a splenectomy in September 2013. So, that's six months after her pancytopenia and bone marrow was done. That was reported as congestive splenomegaly. She then presented to us in Manipal Hospital, Bangalore, in Jan 2014 with severe bone pains and fever. So, we had only this history. The patient did not bring any slides or reports, just a letter, um, because it was a government exchange thing, I think, only from the recording hospital letter with a short case summary. When the patient presented to us in our ER, she had a high WBC count of 29,460, low hemoglobin, and very low platelets. Her peripheral smear showed monocytosis and, uh, shift to left, dysgranulocytosis, toxic granules, and monocytosis. So, we knew that we were not looking at an ALL relapse, but we didn't know what we were looking at. Her LDH was, LDH was high, and alarmingly high alkaline phosphatase was high. All other autoimmune workup was negative. We did a bone marrow aspiration and biopsy, and the bone marrow showed extensive necrosis. We were not able to get any viable cells. However, we went ahead and attempted an immunohistochemistry, which again did not highlight any cells. A PET scan showed a short diffuse metabolic activity in axial and appendicular marrow with multiple mixed lytic sclerotic lesions. This is important because when we discuss the case, we need to know why I'm emphasizing on the PET scan. Multiple mixed lytic sclerotic lesions and low-grade metabolic activity and enlarged bilateral axillary and retroperitoneal nodes. We attempted a flow cytometry on the peripheral blood. We did not show blasts because as of now, we are still looking at the case as an ALL relapse. A CT-guided biopsy from the sacral node, sacral bone, which was a PET active region, again showed necrosis. Actually, a lymph node biopsy was done, which shows her shoulder reactive node. The patient had was doing really badly, and we were stuck. So, then we asked the parents to get all the slides, original slides, and blocks from Mauritius. And patient was started on steroids in the meantime. So, I spoke to the oncologist and I said, in all my, I mean, limited experience over so many years, I have never seen a case and being stuck like that. Can I just have a marrow once more in the time that they get their slide from Mauritius? So, repeat bone marrow was done. We've already lost a month, as you can see, in trying to get her diagnosis. A repeat bone marrow aspiration biopsy was done, which showed of these kind of cells. Can, yes, can you see my pointer? Am I able to? Okay. So, these cells which were extremely granular. This is the bone marrow, extremely granular and convoluted. There was no Auer rods, but I thought that I was looking at promyelocytes. So, once again, I am, I will highlight that, you know, the topic, but till now, I don't know what I was dealing with. So, I thought that these are, they resemble promyelocytes. Of course, it didn't really fit into the history, but that was the best diagnosis I could make looking at the bone marrow. We did a cytochemical MPO, which was negative. We ran the flow cytometry on this bone marrow sample, which of course showed negative cyto MPO, cyto 1798. None of the leukemia-specific markers were positive. Very bright CD117. And all these cells were going into the area which was like close to the eosinophils, and we wondered whether we are really looking at a blast or we're looking or promyelocytes or looking at mast cells. And then we don't do Toluidine Blue. Dr. Tejinder, they will of course take me up on that. We don't do it in hematology, but we requested the histopathology section to do a Toluidine Blue for us, which confirmed that they were mast cells. I had never seen mast cells like these before. We went back and looked at the peripheral smear and then noticed that probably what we were calling promyelocyte shift to left, there were circulating mast cells in the peripheral smear. What we would call 24% monocytes, and what we're calling dysgranulocytosis were, I think, actually all abnormal, uh, mast cells, circulating mast cells. And, uh, so when we had run a flow initially, we were just looking at the blasts to, you know, identify whether this was a relapse ALL, but hadn't concentrated on looking at these abnormal, uh, mast cells on the peripheral blood. A bone marrow biopsy, of course, showed complete replacement, diffuse infiltration with the mast cells. If you can see these nice monocytic convoluted nucleus, abundant cytoplasm, and this is our CD117. I would like you to see this CD117 because I wanted to compare it in the next case. Intensely highlighted mast cells with CD117. And this was a mast cell tryptase, which my colleague who was working with us then, recovered, got it done from her center in the US where she used to work because we didn't have IHC mast cell tryptase here. We called it, uh, mast cell leukemia, and we sent it out for second opinion to JAL at that time. They were in, I think, 2014, they were in SRL, which confirmed that we were indeed looking at, uh, mast cell leukemia. He did a CD2, CD25, CD43. All the tumor cells expressed this and negative for CD34 and pure, just like our flow pattern. So, the clinical course of this patient was very stormy. In the, in our hospital, they managed to get the splenectomy slides, and when we did the IHC, we saw that there was mast cell infiltration here. Bisphosphonate was given IV Zoledronic acid 400 mg over 7 days, followed by imatinib. Her bone pains and thrombocytopenia improved, but a repeat marrow later showed almost 90% of these cells. Alkaline phosphatase continued to be high, LDH was normal. She was discharged in April 2014, but very subsequently, as soon as she reached her home country, she passed away. So, there was this saying by D.H. Lawrence which says that what the eye well, the eye doesn't know when the mind doesn't, but the eye doesn't see and the mind doesn't know does not exist. But in this case, my eyes were seeing something, but my mind didn't know. And there were these hypergranular cells with bilobed or multilobulated nuclei, which I was, which I thought initially on morphology were promyelocytes, but they were promastocytes. They are mentioned in the WHO, but somehow, I think we don't really read them. And the dysgranulocytosis that I was noting were all these abnormal mast cells in the peripheral blood. Also, when you have MPO negative and your clinical suspicion is that you are seeing granular cells, it is good to do a Toluidine Blue. And maybe at that time, I didn't have CD25, but I did have CD2. I could have included it in my flow panel. Now, watch this case. I will discuss the classification of mastocytosis later. Was this case actually a systemic mastocytosis with associated hematological neoplasm? It seems far-fetched because her ALL was many years ago, but if you read the WHO carefully, it says that as many as 30% of systemic mastocytosis cases, there is an associated hematological neoplasm, which is diagnosed before, simultaneously with, or after the systemic mastocytosis. So, we didn't have any molecular workup in 2014. We didn't even have much access to many labs doing NGS. But this was our first case of mast cell leukemia, so to say, that I had seen. I looked very, very carefully at many pictures of mast cells, which shows that they have diverse morphology. These are those abnormal spindle-shaped hypergranular cells. These are the mast cells, which of course, your dense granules can mimic basophils. Here again, some have glass-like morphology which have granules. I kept looking at my marrows, you know, subsequently to see whether I could see mast cells, but I could see only these typical mast cells in one of my marrows, and maybe one mast cell in many high-power fields. Many years passed by. My mast cells were then replaced. They got masked, and the memory was short. And then I said, okay, maybe these cases are again once in a blue moon until April of last year. We had an 11-year-old female with generalized weakness and fatigue, and her main problem was her sudden drop in hemoglobin. She was treated elsewhere in a, a good hematology setup, no response to IV iron, B12. She had received multiple PRBCs, anti-intrinsic factor antibody. I don't know why they had done that, but I'm sure that had high index of suspicion was positive, and she was referred to our hospital for bone marrow studies to rule out HLH. She had come to pediatrics department first. Now, the lab investigations showed in our hospital, hemoglobin of 8.5, despite multiple transfusions, reticulocyte count which was 1.2, her indices were normal, her saturation was very low, B12 was very low despite being treated. All other biochemical parameters were normal. Her HCG was negative. Two peripheral blood smears and thrice consecutively were negative. And her ultrasound showed hepatomegaly of 14.8 centimeters. She was referred to us primarily for a bone marrow. So, this was her bone marrow, and since the clinical suspicion was HLH, we did see a marrow which was diffusely infiltrated by what I thought were histiocytes, and we saw your erythrophagocytosis, RBCs being eaten up. Hemophagocytosis here and there, a lot of cells with debris, and a proliferation of, uh, cells which appear to be histiocytes. Again, here, cells here, some debris here, but she didn't maintain other cell lines unlike the other case, which I had shown you. She did have erythroid cells. Her bone marrow biopsy showed megakaryocyte suppression of other series, but you could see a nice infiltration with these cells which so far we were suspecting histiocytes. So, we didn't run a flow on this case and straight away went and, um, now if you see her CD117 here, it has a distinct pattern, different pattern compared to the case which was a hypergranular mast cell leukemia case. This is her CD68, and her other markers for histiocytes were negative. CD68 of course can be highlighted in mast cells as well. This patient, we diagnosed as mastocytosis. That's all we kept it at because we yet had to do her further investigations. But she then went to CMC Vellore. When she went to CMC Vellore, they did a mast cell tryptase, which was totally normal. Her alkaline phosphatase is phenomenal. This is unusual because when we discuss the classification of mastocytosis, having so much of infiltration in bone marrow with mast cells with a normal mast cell tryptase is an unusual finding. It's not, it's not very, very, I mean, it's not that it doesn't happen, but it's rare. Her PET scan showed diffuse involvement and diffuse metabolic activity in the bone marrow, hepatomegaly, and some small nodes. Now, this is her molecular workup in Vellore, which showed that she didn't have any KIT mutation. This was another, um, surprise to us. So, this was a patient who had low or normal mast cell tryptase in the serum. The IHC wasn't done, and she had no mutation. Her next-gen sequencing by 57 gene panel showed no abnormality. She didn't have KIT mutation. Later, she developed a skin lesion. This was not biopsied, but this is much later. This is also after she was in Vellore. She had COVID as well, so this all delayed her treatment, and her clinical course again, like the previous case, was not too good. She had two cycles of chemotherapy. She received Myleran. A repeat bone marrow showed 20% mast cells, and they went ahead and did her BMT with, with her mast cells persisting because she was, she kept on dropping her hemoglobin. She underwent haploidentical transplant with one of her parents being her donor. She developed VOD, and subsequent to the transplant, had a foot drop, and MRI showed an extramedullary mass. Till as of yesterday, the, you know, the advantage of working in a large hospital is that we, we are still in touch with the clinicians all the time. As of yesterday, she continued to be unwell, and I got her report from her, her relative, saying that even her post-transplant bone marrow biopsy, which was done in December, continued to show, uh, mast cells. So, these were the two cases that we have seen so far, and I, I think I've been doing hematology for more than 25 years now, and I was five years in Tata. I had never seen a case of mast cell leukemia or aggressive mastocytosis like this. So, to give some credit to these mast cells, which have been so far being called only as bystanders and amplifiers and allergies, immunology, and inflammation, they are now serious players because we know that they are derived from multipotent hematopoietic progenitor cells. They differentiate and they move from the bone marrow to the peripheral blood, and then they home in on multiple, in multiple organs. Paul Ehrlich had discovered them in humans. They have been seen for centuries and centuries in other mammals in 1879, and he actually got the Nobel Prize in 1908 on his work in mast cells. First described, of course, by the unique staining properties, metachromatic properties that we call. But today's discussion, we will limit to only the mastocytosis as seen in hematology and hematological neoplasms. As we define mastocytosis as a clonal, clonal neoplastic proliferation of mast cells which accumulate in one or more organ systems. They were first documented in urticaria pigmentosa in 1869, and then the first reported case of systemic mastocytosis was seen in 1930s in France. In the previous WHO, mastocytosis was just a subgroup of the myeloproliferative neoplasms, but now because of its unique clinical and, uh, diagnostic significance, it is a separate disease entity in the WHO. It has to be strictly distinguished from mast cell hyperplasia and mast cell activation states, which are huge in allergy, immunology, and inflammation, but I will not be addressing those cases. We will restrict ourselves to clonal and neoplastic mast cells. The diagnostic criteria of cutaneous mastocytosis, which also I will not discuss, but I will just tell you the classification of cutaneous mastocytosis is either arterial urticaria pigmentosa, which is or maculopapular cutaneous mastocytosis, or diffuse cutaneous mastocytosis, or solitary mastocytoma in the skin. Now, these are characterized by typical histological infiltrates of mast cells, either multifocal or diffuse in a good skin biopsy, and features or criteria which are sufficient to establish the diagnosis of systemic mastocytosis must be absent in cases of cutaneous mastocytosis. So, today's discussion is going to be on systemic mastocytosis, which is then classified in the WHO as either indolent, SMOLDERING systemic mastocytosis with AHN, associated hematological neoplasm, aggressive systemic mastocytosis, mast cell leukemia, or mast cell sarcoma. Now, indolent systemic mastocytosis and smoldering are, you know, they have, they're a separate entity because these patients have good prognosis. However, systemic mastocytosis with AHN, aggressive, and mast cell leukemia, mast cell sarcoma are all advanced systemic mastocytosis. There is a criteria here called B and C findings, which I will discuss later, but suffice it to say right now, indolent and smoldering have very low disease burden. Indolent systemic mastocytosis almost always presents with skin lesions. Now, in systemic mastocytosis with associated hematological neoplasm, it meets the criteria for systemic mastocytosis, but there has to be a criteria for an associated hematological neoplasm, which is most often a CMML or an MDS or an acute myeloid leukemia. However, if you have an isolated node, say a stage one Hodgkin's or an NHL which is totally separate and no other features, that that neoplasm does not qualify along with systemic mastocytosis. It does not qualify to be in AHN. Aggressive systemic mastocytosis meets the general criteria for systemic mastocytosis, but has the C findings, which I will discuss next. Skin lesions are usually absent. Mast cell leukemia meets the criteria for systemic mastocytosis by the, you know, by the definition, you would feel that mast cell leukemia almost always should have circulating cells in the peripheral blood, but that's not always true. Many, many of these cases of the few cases of mast cell leukemia have even less than 10% or no circulating mast cells in the peripheral blood. So, to qualify as it for the diagnosis of systemic mastocytosis, we have major criteria and minor criteria. The major criteria is if you have multifocal dense infiltrates of blood of mast cells or more than or equal to 15 or 15 mast cells in aggregates in the marrow or in an extracutaneous organ, that's a major criterion. In minor, you have four criteria. The biopsy sections of the bone marrow or the extracutaneous organs must show more than 25 plasma cells which are either spindle-shaped or an atypical morphology, or they are immature or atypical in all the mast cells in the marrow. Alone, detection of a KIT mutation is a minor criterion. As you can see that our case didn't have a mutation, so it still, I mean, it's good that it was a minor criterion for us. Mast cells have to have an average expression. Mast cells in the marrow or in the extracutaneous organ express CD25 or CD2, which are both actual not mast cell markers, in addition to other markers. You have to have an average serum total tryptase has to be persistently high, unless in in associated hematological neoplasm where it doesn't qualify, then then tryptase values don't matter. So, if you have one major or one minor, or you have more than or three minor criteria, your case qualifies to be a systemic mastocytosis. Now, I had mentioned B and C findings. These are unique. This kind of findings are unique to systemic mastocytosis because they have, you know, it's a mnemonic. B is says the burden of disease. C, H, C is where there is organ dysfunction and you will require cytoreduction. So, what are the B findings? B findings are the high mast cell burden, which has bone marrow biopsy has more than 30% plasma mast cells, or a very high serum total tryptase, or there is sign of dysplasia or myeloproliferation in the non-mast cell lineage, but the criteria are not definitive to qualify an AHN, or hepatomegaly without impairment of liver function, which was seen in our case where there was hepatomegaly but liver enzymes were normal. C findings are when there is organ dysfunction and cytoreduction is required. There is bone marrow dysfunction or which leads to one or more cytokines. So, if you see our second case presented only with low hemoglobin. She had no other symptoms at presentation. Palpable hepatomegaly, skeletal involvement like our first case, which shows that, you know, you can have all sorts of, I was reading a chapter in an orthopedic article which says that you can have every possible bone involvement, not bone marrow, bone involvement in systemic mastocytosis. Palpable splenomegaly with hypersplenism, also seen in our case one. And malabsorption with weight loss due to GI symptoms. GI symptoms are a majority of the cases of systemic mastocytosis have very, very strongly GI symptoms. So, once again, to just go through the criteria, I don't want to eat into my colleagues' time. I have to work with them for the rest of my life, I think. So, systemic mastocytosis, um, can be divided into indolent or smoldering or aggressive depending on the B O C symptoms like I have described earlier. Now, so far, we spoke of only CD2 and CD25, and the new proposals which have come to update the guidelines and the criteria for assisted diagnosis of systemic my, uh, mastocytosis. CD30 has become, uh, an important marker because it's seen in neoplastic mast cells in the majority of patients which have either indolent or advanced disease by flow and IHC. We did put up CD30 in our second case. Now, in the histological subtype of mastocytosis, there is something called a well-differentiated morphology where you have these round mast cells which are intensely granular, and in the well-differentiated morphology, often you don't get CD2 or CD25, but they have almost always expressed CD30. There have also been cases who are being treated with Rituximab, which is anti-CD, monoclonal antibody against CD30. So, CD30 has now been proposed to be a new addition to the existing minor systemic mastocytosis criteria. In the molecular genetics, we know that KIT mutations, more than 90% of adults with systemic mastocytosis have KIT mutations. The commonest KIT mutation is what we see, the D816V, in which adenine to thymine base substitution happens, which results in an aspartic acid to valine change in the protein. Now, this mutation leads to constitutive stem cell factor independent activation, which leads to downstream signaling, and there is a proliferation, growth, proliferation, or growth, or or giving a survival advantage to the mast cells. However, there are many other KIT mutations which can also be seen. We know that more than 80 to 90% have D816V, but there are a lot of other mutations which can be seen in cases of systemic mastocytosis. Just a small, uh, illustration to tell you how the KIT mutation works. This is a normal hematopoietic multipotent hematopoietic stem cell. This is normal. These are two abnormal stem cell factor here, which is a ligand for KIT. The, this is the multipotent stem cell goes into the marrow, everything is fine. Stem cell factor is still here. It matures into a committed mast cell progenitor, mast cell, which is still granular in the peripheral blood, homes onto other tissues, becomes granular, and then secretes either tryptase or chymase or any of the other mediators. And this is a normal mast cell, which is when it's mature, it's granular. There are two pathological conditions here. If you see here, the multipotent stem cell is normal, but somewhere along the line, before it is, you know, it comes out into the peripheral blood, you acquire a D816, you know, the stem cell acquires a D816 mutation before it's getting committed to a mast cell progenitor. It may also acquire additional mutations. So, additional mutations, and that's why the importance of doing NGS are very important because they, they decide whether you have a lower allele burden or a high allele burden or aggressive mastocytosis. So, cells which acquire D816 mutations later after becoming committed mast cells have a lower allele burden, and these are the indolent ones. However, when all the first hit mutations and the D81816V occurs immediately, even before they become committed mast cell progenitors, these are the aggressive mastocytosis cases. Now, there are 5 to 10% patients which are negative. And why are they, which are the scenarios in which they can be KIT negative? These may be patients which are truly actually KIT positive, but they had a low mast cell burden, and therefore, you need a very, very sensitive assay. Sanger sequencing may not be the right thing to do to pick up these, uh, mutations. And if this fails in a case where you have a strong suspicion of cystic systemic mastocytosis, perform it again on the peripheral blood. Then the patient may be truly a wild type KIT, doesn't have mutation. All the patients may carry other KIT mutations. So, these are the cases to really truly call them KIT negative. First, we have to be sure about the technique we are using, the sample we are using, before we declare them KIT negative. A quick, uh, mention of something called myelomastocytic leukemia. I haven't seen a myelomastocytic leukemia, but with your Balouns group, the Austrian group, which has worked a lot on mast cells, have classified something called have defined myelomastocytic leukemia as a leukemia which has myeloblasts as well as mast cells. So, these are not AML with AHN, but they are myelomastocytic leukemia where skin lesions are absent. They, the tryptase will be normal or low, and they have circulating myeloblasts as well as mast cells. They do not have KIT mutations. So, just to summarize the diagnosis of systemic mastocytosis, this is what I found in one of the ASH publications. A new toolkit, mastocytosis, of course, along with clinical history, has to be diagnosed with bone marrow with IHC, with imaging, with organ biopsy, with the KIT mutations, with allele-specific oligonucleotide quantitative PCRs, not only Sanger sequencing, a review of the peripheral blood, and of course, expert clinical inputs. For more details on mastocytosis, there's an entire book on mastocytosis, and this is a very important, um, update which has come out last year, which is the EHA publication from this Austrian group, which has I, these are the ones who wrote the chapters in the WHO, which have updated the diagnostic criteria, and maybe in the next WHO, we have a refined criteria for diagnosis of mast cell disorders. Thank you very much. This is all about mastocytosis.

Thank you, Dr. Swati. Wonderful review and beautiful cases. So, we want the second speaker, Dr. Roshini Shekhar, on TAP.

Hi, and good afternoon. At the outset, I'd like to thank Agarwal for inviting us and the Mumbai Hematology Group. It's a great honor to be here. My topic today is on TAP. I'll just share my screen. Is my screen visible? Yes. Thank you. Um, so it's quite a simple topic, but it's not too commonly seen, so we thought we'd discuss it today. So, we, I start off with my case. We had a case referred to us at a pediatric hospital here in Bangalore. It was a three-day-old male baby, suspected Down syndrome, hadn't been confirmed. And the only complaints that the clinician called and told us was that the count was increased and the platelet count was on the lower side. No history of jaundice, no history of rash, organomegaly. LFT, RFT were normal. So, we said, send us the smear, let's take a look at the slide. So, we got the CBC, and the count was increased, 49,360. Hemoglobin is 14.6, and platelet count was 31,000. So, obviously, the first thing we do was, we made a peripheral smear, and straight away, we saw that there's a significant increase in blasts. So, we can see that this is the low power view. We have a higher power which is a bit easier to see. So, you can see that all of these cells here, these are all blasts. Increased N:C ratio, irregular chromatin, opened up chromatin, some prominent nucleoli. So, immediately, we called it acute leukemia. And then we said, would you like us to do a flow? So, they said, please go ahead. So, we went ahead with the flow for this patient. So, so over here, um, so the blasts, we get to them in the CD45 positive, negative sites, scattergram area. Uh, they expressed, uh, stem cell markers, CD34. They were negative CD19. Uh, there was some dim CD7. This is not actually a very specific finding in this case, but anyways, we had a dim CD7, and we had a negative cyto CD3, and there were some dim CD33. Uh, MPO was negative, slide MPO was also negative. Uh, the patient also had DR positivity, some CD117 positivity. CD14 was negative. And over here, so this was where it got like a little more testing. We are also putting a CD41 and CD71, which are the two glycoprotein markers, and we saw some dim CD71 and a small subset did appear to show CD41 positivity. CD56 was negative, and CD15 was negative. So, now, given this history of Down syndrome, three-day-old baby, increased blasts in the peripheral smear, crossing 20%, and the expression of stem cell markers, myeloid markers like CD33 and CD117, plus also platelet glycoprotein markers, and the absence of any other lineage-specific markers, we decided to call this, uh, TAM, or Transient Abnormal Myelopoiesis. So, this patient subsequently, the sample was sent for karyotype, which confirmed Trisomy 21. And very interestingly, this was sent for sequencing to Metagenome, and this patient came back with a positive GATA-1 mutation. So, this basically solved our dilemma. This was definitely a case of TAM. This patient also showed JAK3 mutation, which is not very specific. It just, it can be seen along, um, in Down syndrome patients. So, coming to the discussion, um, so TAM is one of the, it comes under the category of myeloproliferations associated with Down syndrome. So, there are two entities in this. One is TAM, which is Transient Abnormal Myelopoiesis, and the other one is Myeloid Leukemia associated with Down syndrome. So, this has a separate category in the WHO. 2008 was I think included as a separate entity on its own. So, Down syndrome is one of the most common causes of, one of the most common chromosome abnormalities, and these patients are at a, it's known that there are increased risk of developing leukemia. Oddly enough, though the ratio of, uh, lymphoplastic to myeloid leukemia in these children is one is to one is to 0.1, 0.2, so they have a higher incidence of acute myeloid leukemia. So, there's a 150-fold increase in AML in these patients, out of which, um, the entity that's acute megakaryoblastic leukemia accounts for 70% of these cases. We'll also talk a bit more about that later. And about 10% of patients with Down syndrome develop something called TAM. So, why is it called Transient Abnormal Myelopoiesis? It's because it, it's like a, it's indistinguishable from an acute leukemia when you first see it because it's more than 20% blasts, it looks like a straight-looking leukemia, but it may resolve on its own within a few weeks to a few months. So, if we look at the incidence and evolution, this might make a little clearer for the students. So, if we have, so we start off with a patient who has Down syndrome or Trisomy 21. Incidence is not too common, about one in 700. Now, if this patient doesn't have any GATA-1 mutation, so the GATA-1 mutation is sort of the crux of this whole development of TAM and then myeloid leukemia with Down syndrome. If you lack the GATA-1 mutation, you don't develop TAM, and then you would therefore don't develop myeloid leukemia with Down syndrome. So, about 70% of these patients lack the mutation, and therefore do not develop that. Now, in the remaining 30%, and if they have the GATA-1 mutation, again, either you could have a large clone or you could have a smaller clone. If you have a smaller clone, you may not actually have an increase in blasts, but you have the GATA-1 mutation. So, in these cases, you would call it silent TAM. This is about 20% of patients. Now, in this, perhaps the clone itself, which is the mutated clone, it may go extinct, and you may never develop, uh, Down syndrome myeloid leukemia, or sometimes these patients acquire additional mutations, and then they may later develop, uh, myeloid leukemia with Down syndrome. So, this leukemia does not, uh, regress on its own. This would need treatment also. So, now, so this is if you had a small clone where you didn't have a significant increase in blasts. If you, however, had a larger clone and you had more than 10% blasts, this will be picked up most likely by a hematopathologist or by your clinician, and this will then become clinical that this is about 4%.

To ten, they say about seven percent of patients develop this. So again, similarly, if these, if this clone, this clone of GATA1 unmutated cells just automatically goes extinct, these patients do well and they don't develop any secondary leukemia later on. However, some of them again may develop additional mutations, which then leads them to develop myeloid leukemia, which is, this is a non-spontaneously regressing kind, out of which about, there's a mortality rate of about 20 percent, but about 80 percent do well. So there's an 80% survival rate. This leukemia is also very sensitive to chemotherapy, so it overall does much better than AML in patients without Down syndrome. And over here, in clinical term, about 20% or less don't do well, and they may expire in the stage of clinical term itself.

So, coming to the disease mechanism. So in 2002, Crispín and their group, they discovered this GATA1 mutation and they made this association. The GATA1 mutation leads to the development of TAM, and then later on, megakaryoblastic leukemia. So the current hypothesis is that TAM arises in the liver, and that's one of the reasons why they say it spontaneously disappears. Because as hematopoiesis switches over from the liver to the bone marrow, the clone itself of these malignant hematopoietic cells goes down because the bone marrow is not producing all the cells.

So if we see over here, you have a hematopoietic cell with trisomy 21, which then has the GATA1 mutation and leads to your transient leukemia. Now, this may go spontaneously into remission, or you may have a latent transient leukemia clone, which then later on acquires additional mutations and then may develop a secondary megakaryoblastic leukemia. In between these stages, you may have an almost MDS-like phase where the patient normally presents with cytopenias.

So the leukemic blasts of Down syndrome AMKL harbor mutations of GATA1. GATA1 basically encodes a hematopoietic transcription factor. It's based on the X chromosome. Most of these mutations are within exon two, and they result in the expression of a shortened, a truncated mutant protein, which leads to all the, which lacks the amino-terminal transcription activation domain.

So now, when to call your TAM? This is also a bit of a controversy. Not many people can agree when, at what percentage. Even the WHO says an increase in blasts, but it doesn't define the cutoff of which you would actually call a TAM. The Children's Oncology Group, so their study, they based their criteria on detection of non-erythroblasts of any number in the peripheral blood or in organs of newborns and infants less than 90 days with trisomy 21. They also require that there was a second blood sample or additional findings of more than 5 non-erythroblasts. The BFM study of 0.1.9.Jester, that more than 5% myeloid blast in the peripheral blood or the bone marrow aspirate within the first six months of life can be diagnosed as TAM. The Oxford Imperial Down Syndrome Cohort study studied a few more. So these two other groups studied about 150 patients. The Oxford group studied around 200 newborns, and they looked at blood counts, blood morphology, percentage of blasts, clinical findings, and GATA1 mutation status. Also, in this, they found that almost all newborns actually had circulating blasts. So you would have to, so just saying the presence of blasts may be insufficient for calling it TAM. They also then sequenced most of these patients and they found that GATA1 mutations were seen in about 8.5% of patients, and all of these had more than 10% blasts by morphology. In addition, 20% they found a GATA1 mutation, but they didn't have any clinical features or hematological features. So they labeled these as silent, wherein you have the GATA1 mutation, but you lack clinical features and the morphological features. They also noted that if you had more than 20% blasts, you invariably had a GATA1 mutation, and a diagnosis of TAM was very easy to confirm. The group finally concluded that more than 10% blasts was their threshold for diagnosis and management of TAM.

So the spectrum of clinical features and TAM, this ranges from almost asymptomatic, like the patient we saw had next to no symptoms, it just came based with high count. But some patients develop quite severe, life-threatening symptoms. Also, most patients are seen in the first few weeks of life. Some of the common symptoms are hepatomegaly, splenomegaly, jaundice, plus other clinical symptoms, thrombocytopenia, and leukocytosis. Some of the more life-threatening symptoms that we can see are hydrops fetalis, hyperleukocytosis, liver failure, cardiopulmonary failure, and a lot of patients present with pericardial or pleural effusions. Infiltration of solid organs is not so common. It's generally seen in the liver, spleen, marrow, and the skin. Hepatic inflammation is seen commonly as the cell of origin is in the liver, and liver fibrosis is seen in about 7 to 15% of cases and it's an unfavorable prognostic sign.

So coming to the lab features. So TAM is seen as categorized by presence of blasts in peripheral blood. Again, I've not mentioned a cutoff because it's a bit controversial, but more than 10% is what the Oxford group concluded. So typically, these blasts in TAM are called megakaryoblastic. So over here, we have a picture where you have this sort of blebbing from the cytoplasm, this basophilic cytoplasm. But from what we've seen, and we've not seen, we've seen hardly about two, three cases, the morphology is not consistent. In the case that we had that I've shown earlier, we did not see this blebbing with the megakaryoblastic morphology. So that isn't, like it may not always be seen. Leukocytosis is common, and some patients may present with hyperleukocytosis, which is more than one lac count. Hemoglobin patterns can be decreased, but not necessarily severely reduced, and some studies say that they can be associated basophilia. Also, I would just like to point out here that acute megakaryoblastic leukemia, which is under the AML NOS category, if they've mentioned over here, if this occurs in a patient with Down syndrome, then it immediately gets shifted to the myeloid leukemia associated Down syndrome category.

So the immunophenotype of TAM. They normally have CD34 positivity, CD117, myeloid markers like CD13, CD33, CD41, 42, 36, 61, and 71 are often seen. These are the platelet-like protein markers, and they may show avid expression of CD7 and CD56. So I won't be discussing MDS, but just for comparison, in MDS, the immunophenotype is very similar. So this is the leukemia that develops later, normally within the first four years of life after the TAM has regressed. So in these patients, the immunophenotype is similar. However, CD34 is negative in about 50% of those cases, and 30% of these cases are negative for CD56 and CD41.

So other causes of peripheral blood blasts. This is not commonly seen, but if we should at least think about these conditions. So a severe infection, sepsis, congenital infections, TORCH syndromes, sometimes in hemolysis when there's a lot of stress erythropoiesis, you may see some circulating blasts. And although the incidence of all in Down syndrome, as we discussed, is extremely low.

Bone marrow examination. So in the two patients that we've seen, bone marrow examination was not done, and it's commonly not done because normally the percentage of blasts that you see in the marrow may actually be less than the peripheral smear because the blasts aren't coming from the marrow. But if, so it's not routinely recommended. At least in the two cases we've seen, it was not done at all. But if it is done, so this is not our case, this is a picture I've taken from a textbook. But if it is done, you can see. So this is showing increase in blasts. These are these large abnormal looking cells over here, and then you have a few erythroid cells in between that, and I think some a couple of mature myeloid cells also. But according to literature, you may see abnormal megakaryocytic maturation and dyserythropoiesis.

Other lab investigations. Coagulation tests may be abnormal in over 20% of cases. DIC is seen in about 10%. The TIC is generally seen in the most severely affected patients and it's a significant risk factor. So some of the other investigations to be done are your PT, PTT, fibrinogen, D-dimer, LFT, and RFT to look at liver status and electrolytes. In terms of molecular studies, a karyotype to confirm trisomy 21, and if the patient can afford and if it's possible, then sequencing to look for the GATA1 mutation. So if we see over here, this is the normal sequence in exon 2. However, in this one, exon 2 is mutated. And the normal protein, this is the mutant protein. This section over here has been knocked off. It's truncated, it's a smaller protein.

So now, when would you actually suspect TAM? So it's normally, so if you have a patient with Down syndrome and you found blasts on the peripheral smear and abnormal counts, then that's one of your first clues. This you could follow up with flow cytometry to confirm the immunophenotype. So if you're looking and you're seeing CD41, CD71, or CD42, that also helps in your diagnosis. This would obviously then be followed by cytogenetic analysis to confirm trisomy 21, and the gold standard would be the GATA1 mutation sequencing. In addition, if you have young infants less than three months who have symptoms like hypoleukocytosis, hydrops fetalis, ascites, hepatosplenomegaly, abnormal LFTs, liver failure, unexplained effusions, these patients should also be worked up and investigated at least to rule out TAM.

When to treat? So again, this is not the most standardized. Again, but overall, I think most groups have come to some kind of consensus. So a majority of patients, TAM resolves spontaneously. That's about 40%. They resolve on their own, they don't need any intervention. In the Children's Oncology Group, they saw that blasts disappeared after a mean of 36 days. The range was quite large, due to 126 days, and all signs of TAM, including the hepatomegaly, had dissolved after about 50 days. So low-risk patients do not require intervention. However, patients who are intermediate or high-risk, so high-risk everyone agrees definitely need intervention. Intermediate, it depends from clinician to clinician. So in this group, they divided into low-risk, intermediate, and high-risk patients. So low-risk, which was about 40% of all patients, there was no hepatomegaly, and there was no hepatic dysfunction, no life-threatening complications. So these patients were only observed. In the intermediate risk, there was hepatitis plus evidence of non-life-threatening hepatic dysfunction and no life-threatening complications. So here they recommended that you start very low-dose cytarabine therapy. So something unique about these patients is that they are very sensitive to this chemotherapy and they respond very well once you start the treatment. And high-risk patients, which is about 20%, these are patients where you have cardiorespiratory compromise, pericardial pleural effusions, more than one lac count, severe life-threatening hepatic dysfunction. In these patients, they recommended aggressive supportive care along with cytarabine therapy. The BFM group identified four risk factors that they said was associated with increased probability of early death, and these patients definitely demonstrated benefit from low-dose cytarabine. So patients with ascites, preterm delivery, hyperleukocytosis, and bleeding manifestations. The pediatric oncology group similarly said that these were their criteria for treatment, which is patients who had these life-threatening symptoms. So multi-organ failure, increased WBC count more than one lac, beyond the umbilicus or causing respiratory feeding compromise, hydrocephalus, pleural or pericardial effusions, renal failure, or DIC.

So this is the next step. So now the patient has TAM, what happens next? In most cases, as we said, they resolve on their own within several weeks to three months. But in about 20 or 30% of cases, within about one to three years, normally within less than five years, they develop non-remitting or non-spontaneously retesting acute megakaryoblastic leukemia. So what the, what the consensus seems to be is that while you have your GATA1 mutated TAM clone, if this stays on and it requires additional mutations, so like a CTCF or a JAK2, JAK3, MPL, any additional mutations, this then leads to the development of this myeloid leukemia associated with Down syndrome. However, this also has favorable prognosis with good response to chemotherapy.

So BGS put out a couple of guidelines a few years ago where they said that, so where they've given a couple of recommendations for diagnosis and treatment investigation of TAM. So they said that transient leukemia should be defined as the presence of a GATA1 mutation along with a blast percentage of more than 10% in a patient who has Down syndrome. All neonates who are known or have a high suspicion of Down syndrome should be examined for features suggestive of TAM. So to look for organomegaly, cholestasis, hepatopathy, skin rash, effusions. And in addition, in the first three days of life, they should have a full blood count and a blood film examination with assessment of peripheral blood blast count. And any child who, even though if they don't have a peripheral blood blast percentage performed at the first three days of life, or if there was significant IGU where the blast counts may be suppressed, they should still be considered at risk of clinical problems of TAM and they should be monitored for the next one to two months of life.

So just to summarize, if you have a neonate with suspected or confirmed Down syndrome, you start off with a blood film and a peripheral smear for blast percentage, followed by genetic testing to confirm trisomy 21. Clinically examine for features of transient leukemia like organomegaly, hydrops fetalis, effusion, skin rash, organomegaly. And if the patient has blasts more than 10% or clinical features of TAM, so if the patient doesn't have more than 10% blasts or clinical features, then it seems to be unlikely that it will be TAM. However, if you can, I mean, if the patient can afford it or it's possible, a GATA1 mutation may still benefit because it may inform you about future risk of developing MDS. If the patient does, however, though have more than 10% blasts or clinical features of TAM, then look for evidence of life-threatening syndromes, symptoms, so like multi-organ failure, hypoleukocytosis, hepatopathy, organomegaly, hydrocephalus, effusions, DIC. And send the sample for GATA1 mutation. If the patient does have life-threatening symptoms, then consider starting treatment. If the patient doesn't have any life-threatening symptoms, then monitor until counts recover, and they suggest monitoring for liver function also. And if the patient later on shows evidence of relapse or counts that begin to increase again, consider repeating the GATA1 mutation again to confirm whether the patient has now progressed to myeloid leukemia with Down syndrome. Thank you. Thank you so much, Dr. Roshni. That was fantastic, almost complete review of the subject with the case presentation. We go to the third presentation by Dr. Vishwat on BPDCN. Hello everyone, good afternoon. Is my screen visible? Yes. So before I begin, I would like to thank Dr. Mohan Hardikar for inviting me to speak on plasma blastoid cell neoplasm. So I'll be starting off with one of our cases and then proceeding to give an overview of the disease with a special emphasis on the laboratory aspects. So the index case is an 89-year-old male who was referred to our hematologist at one of our hospitals from a psychiatric care facility. He presented with fever, dyspnea, and on lab investigations, he was found to have thrombocytopenia. And the peripheral smear which was examined at a local unit showed 60-14% blasts with elevated LDH. So with this background, with the clinical suspicion of acute leukemia, he was referred to the hematologist, and the hematologist sent the peripheral blood sample to our unit for flow cytometry for acute leukemia and whatever history is mentioned here, this was what the history was provided to us, and there was no physical examination finding which was mentioned in the test requisition form. Unfortunately. So with this, we went ahead and did a hemogram which showed anemia and thrombocytopenia with a preserved total leukocyte count. The analyzer also picked up presence of nucleated red blood cells in the peripheral blood. The peripheral smear at our hospital showed 29% blasts. This is a representative image. Sorry for the poor quality, this was taken from an archival slide, and as you can see, they showed a blast which represents lymphoblasts without any specific morphological indicators to point towards any specific type of leukemia. We did a cytochemical stain which was negative, and it was signed out as acute leukemia, and flow cytometry was requested. So we went ahead and did the procedure for this on the peripheral blood sample. So this is the leukemia screening tube which we initially did. The orientation tube, as you can see, that in this tube, we were unable to find any lineage-specific markers in this particular case. So CD3 was negative, CD19 was negative, cytoplasmic MPO was also negative, and there was absence of CD34. CD38 was positive, and there was a dim expression of cyto 79a, CD7 very dim, nothing specific. So after the screening tube, we were like wondering what could this be? Could it be an acute undifferentiated leukemia or something else? So we tried to do a mixture of other markers including myeloid, T cells, and B cells. So we, in this mixture of markers which we put up, we found that these cells express HLA-DR with bright CD56. They were CD117 positive. Monocytic markers of CD64 was negative, CD15 was negative, CD10 was negative, and we noted that there was expression of CD123 with a variable expression of CD4, CD5, and CD8 were negative, and CD56 was CD16 was also negative. So the NK cell markers were negative, myeloid and monocytic markers were negative. So only HLA-DR, CD4, CD56, and CD123 was positive with a variable expression of CD36. So the positive markers were variable CD4 expression with 56, 123 positivity, HLA-DR, 117, 38, 36 dim CD7, and cyto 99 equals 20. All the lineage-specific markers were negative, which are highlighted in blue, except for dim expression of cyto 79a. So the report was signed out as immunophenotype compatible with blastic plasmacytoid dendritic cell neoplasm. Unfortunately, we did not have any subsequent follow-up for this case. So I don't know whether this patient had any other manifestations of this particular disease. So with this background, I'll move ahead with the discussion. So blastic plasmacytoid dendritic cell neoplasm was the initial reports of this disease started in the early 90s, early 1990s, with the first case being described in '94 by Adachi, who described this entity as in a Japanese 67-year-old Japanese patient with skin lesions and peripheral blood blasts as a high expression of CD56 in a patient with cutaneous CD4 positive lymphoma. Subsequently, other cases were reported, published in literature, and in '95, Brodie et al. called this entity as acute agranular CD4 positive natural killer cell leukemia. This was possibly because of the CD56 expression. In 2001, WHO classification, this particular entity was classified under mature T cell and NK cell neoplasms and was labeled as blastic NK cell lymphoma. In 2002, Peto et al. described the cell of origin and they termed its entity as a granular CD4 positive CD56 positive hematodomic neoplasm, which is possibly arising from plasmacytoid monocytes which were CD56 positive, so possibly of plasmacytoid cell origin. Subsequently, in 2008, the WHO classification put this entity under AML and related precursor neoplasms and changed the terminology to blastic plasmacytoid dendritic cell neoplasm. So the current WHO moved this disease out of the category of AML and neoplasms and has made it into a separate category. However, still the terminology remains the same. So in this next WHO classification, we are yet to see how this disease, whether the terminology will change or not. So since the first disease, the first report of this disease was published, there has been a spurt of publications because this disease entity has been recognized more, and also it has been included in the WHO classification. That's why subsequent to 2008, there's been a growth in the publications indexed publications in PubMed. So this disease is a clinically aggressive tumor which is derived from precursors of plasmacytoid dendritic cells and accounts for less than 1% of all hematological neoplasms. So it's a very rare entity, and the estimated incidence is approximately 1000 cases annually in the West, with a particular male predominance of cases. Most patients present in the fifth or the sixth decade of life, however, there are reports that this can occur in any age group, including in children. There is a possible bimodal distribution with a peak in the younger age group, that is in the less than 20 years, and the second peak in the after the age of 60 years.

So a little bit about the precursor or the cell of origin, which is the plasmacytoid dendritic cell. It forms a part of the immune system, and this particular cell specializes in the production of type 1 interferons. The PDCs are derived from the monocyte-macrophage precursor, which differentiates into the common dendritic cell precursor under the influence of CLIP3 ligand. There have also been reports of a common lymphoid precursor giving rise to PDCs. This could explain the expression of T cell and B cell, some T cell and B cell markers in plasmacytoid dendritic cell neoplasm, which I will discuss subsequently.

Clinically, BPDCN cases present. Majority of the cases present with skin lesions. This can be in the form of isolated papular nodules or blue-like papules, or this can be in the form of disseminated plaques, like we can see in this particular image, that you can see a plaque-like purplish areas or a single large nodule. There are reports in literature where patients have presented without any skin lesions, although those are rare, and half of the cases can present with bone marrow or peripheral blood involvement and lymph node involvement. The leukemic presentation of this disease usually precedes the skin lesions in majority of the cases. Around 50% of the cases can show involvement of liver, spleen, and CNS. Some of the cases of BPDCN have been reported to be associated with or developed into other myeloid neoplasms, most commonly is a chronic myelomonocytic leukemia, also MDS or AML. So this possibly indicates that there is some common genetic basis for the development of these particular neoplasms.

So this particular data which I'm showing, this was presented in last year's ASCO by Dr. Robert Okami, who's from UCSF. His team analyzed nearly 300 PubMed articles using computational pathology and they passed all that data through machine learning and came up with the anatomic distribution of disease in BPDCN and found that majority of cases, around more than 80%, have skin involvement, which most of the lesions being present on the torso or the back and face. And nearly half of the cases presented to them with lymphadenopathy, and bone marrow involvement are also common in nearly two-thirds of the cases. So this is a data from really whatever they could analyze from 284 PubMed articles. So the median age of all the cases around 65 years without a particular male preponderance. And what they found was that most of the cases had a normal or preserved WBC count with a low peripheral tumor burden and low platelet count. So thrombocytopenia was a predominant hemogram finding in most of the cases in published literature.

Coming to the morphology. As you can see from a panel of photos here, the typical morphology resembles a plasmacytoid appearance. The reason why it's called plasmacytoid is because of the presence of frequently eccentrically located nucleoli. And most of the cells have a blast-like morphology. Either they resemble lymphoblasts or myeloblasts. Nucleoli may be present, which may range from one to several, and the cytoplasm is usually gray to blue and it's devoid of cytoplasmic granules. So this was one of the reasons why it was previously called by some of the authors as an agranular hematodomic neoplasm. Some of the cells may show large pseudopodia and microvacuoles in the peripheral blood and bone marrow smears. So this is the typical morphology which is opened up chromatin with this pseudopodia which can give rise to a plasmacytoid appearance. These cells are usually negative for MPO, CA, and NSC cytoplasmic stains, and Ki-67 proliferation index can usually be high. It can range from 20 to 80%.

So this is the panel of photographs showing the morphological spectrum in BPDCN, and this is from the French BPDCN group, and this was published in the How I Treat series. So this is the typical morphology which you can see. I hope people can see my pointers. So granules can be present. There can be presence of cytoplasmic vacuoles, or they can resemble lymphoblasts. They can resemble monocytic cells showing cytoplasmic indentation, sorry, cytoplasm calculations, nuclear indentations, or the typical plasmacytoid morphology. So though there is a huge heterogeneity in the morphological spectrum, there is no specific whether we can say this is a myeloblast or a lymphoblast. That is why whenever there is absence of lineage-specific antigens in your flow, you should always look for this particular disease, even though the morphology may look like an ALL or AML, and you may not find those particular specific markers in flow.

So coming to the cutaneous infiltrates. So the BPDCN tumor cells are usually non-epidermotropic. They spare the epidermis and at nexus. So as you can see in this low-power microphotograph, the epidermis is spared, and usually it extensively involves the dermis with extension into the subcutaneous fat. Lymph nodes are usually diffusely involved with inter-follicular areas and medulla being called, whereas if B cell follicles are often spared. In the bone marrow biopsy, the infiltrate may be either mild interstitial, being detected only on IHC, or they can be massive replacement. So the residual hematopoietic tissue may exhibit dysplastic features, especially in the megakaryocytes. So in case of mild interstitial infiltrate where it may be difficult to pick it up on H&E sections, PDC-specific markers such as CD303 may help in picking up these particular cells, and also this particular marker can be used as an MRD marker on bone marrow sections to pick up because this is a PDC-specific marker.

Majority of the cases of BPDCN have CD4, CD56, and CD123 expression. Apart from this, other PDC-specific markers are also expressed, which include CD303, which is a blood dendritic cell antigen 2, CD304, which is BDCA4, T cell leukemia antigen 1, CD2 associated protein, SPY B, human max virus resistant protein 1, or also known as MXA, and TCF4, which is a transcription factor 4, and CD68 expression also can be seen. So among these, the ones highlighted in blue are the PDC-specific markers. So as I mentioned earlier, that all lineage-specific markers which are described by WHO for lineage assignment are negative, that includes cytoplasmic CD3, 79a, CD22, and lysozyme. Some cases have been reported where there is expression of these lineage-specific markers. However, these are all dim expression and seen in very rare cases that include cytoplasmic 79a and CD3 expression. Cytoplasmic 79a, as I had shown earlier, that it was seen in our case also, although the expression was dim. So the current WHO says that the absence of any lineage-associated antigens together with positivity for CD4, CD45RA, CD56, and CD123 is considered a unique phenotype virtually pathognomonic of BPDCN. So similarly, findings were also seen in the French BPDCN publication, which said that most of their cases expressed CD4, CD123, HLA-DR, and TCL1, and there was frequent expression of CD56, CD304, CD303, CD36, CD38, and CD45RA. Along with this, they also found that the majority of the cases can show expression of at least one T cell lineage marker, the most common being CD7, followed by CD2. So CD7 dim expression was seen in our case also. Around 47% of their cases also showed expression of one myeloid marker, and the most frequent being CD33, followed by CD117. CD117 expression was seen in our case. B lymphoid markers will also be seen, and this most frequent being surface CD22, followed by cytoplasmic 79a, which is in approximately 7% cases. TdT expression also can be seen in these cases, approximately 15% of the cases can show TdT expression.

So with this background, the differential diagnosis of BPDCN. This includes AML with or myeloid sarcoma with monocytic differentiation. These cases can show expression of CD56 and CD13, causing confusion with BPDCN. However, other lineage-associated markers of myeloid lineage will be present in this particular entity. And when it comes to tissue sections, that is skin lesions, leukemia cutis can also show TCL1 expression, or some skin lesions in CML cases have been reported to show CD56 and CD123 expression, causing confusion with diagnosis of BPDCN. The cases with expressed cytoplasmic CD3 and TdT can cause confusion and overlap with TLL or lymphoblastic leukemia cases. The other differential diagnosis include extranodal NK/T cell lymphoma, aggressive NK leukemia, and the exclusion, which is acute undifferentiated leukemia. So especially in the tissue sections, these histological sections, the differential diagnosis include extranodal NK/T cell lymphomas, cutaneous T cell lymphomas, and myeloid sarcoma looking acute myeloid lineage. However, there are certain specific differences between these entities and BPDCN, which help us differentiate it from BPDCN. So the extranodal NK/T cell is a lymphoma that usually, as the name itself suggests, that it's a nasal type. The most common location is nose, nasopharynx, and palate, and the lesions usually show destructive kind of a pattern with feminine necrosis and involvement of vascular walls. And tissue sections, the cutaneous T cell lymphomas usually have epidermotropic lymphoid infiltrate, and they show monoclonal TCR gene rearrangement, which can be seen. So under this category, you can have cases of mycosis fungoides with CD56 expression, which can be confused with BPDCN, or cutaneous ALCLs. Rare cases of cutaneous ALCL, which can show CD4 and CD56 expression. However, most of these cases will have epidermotropism, which is not seen in BPDCN, and which is non-epidermotropic. Apart from that, the immunophenotype and EBV association is also strong in case of extranodal and cutaneous, which helps us separate it from BPDCN. So that was the differential diagnosis on tissue sections. So this should be kept in mind whenever we are seeing histopathological samples or even when these particular entities show leukemic involvement, they can be confused with BPDCN.

A little bit about how to differentiate benign PDCs versus neoplastic PDCs. Benign PDCs, as I mentioned, can show the PDC-specific markers such as CD303, TCL1, CD2 associated proteins, and all the lineage-specific markers and immaturity markers will be negative. Normal circulating PDCs can show weak positivity for T cell markers such as CD2, CD5, CD7, CD33, or CD56. So the difference between the benign cells and the neoplastic cells is that the neoplastic cells usually have a high index. They express one or more normal PDC markers, usually in high intensity compared to the normal PDCs. There is lack of any lineage-specific markers here also, and they commonly express CD4 and CD56. So CD56 expression is one of the key points differentiating from normal benign PDCs, where the CD56 expression can be weak, whereas here it will be usually commonly expressed in majority of these cells, and they'll be strong expression of CD56 in the BPDCN. Variable expression of CD2, CD5, CD7, CD33, and CD36, and TdT can be present, as I mentioned earlier.

So another entity which we should be aware of and this should be distinguished from BPDCN is an entity called as mature plasmacytoid dendritic cell proliferation, MPDCP, which can be present in association with other myeloid neoplasms. Here, the proliferating cells exhibit a similar immunophenotype compared to the normal PDCs. However, CD56 expression, as I mentioned, is negative in most of the cases, or if it is present, it shows focal or weak positivity, and Ki-67 index is low. So this table summarizes the differences between the MPDCP and blastic plasmacytoid dendritic cell neoplasm. So in BPDCN, there is CD68 expression is usually negative or in the form of a single paranuclear dot on IHC, and there will be frequent expression of CD56, CD7, CD33, and TdT, and granzyme will be negative. So this should be kept in mind whenever we are seeing plasmacytoid dendritic cell proliferations in tissue sections as well as in circulating peripheral blood.

So this is just a representative case which was a case of MDS, multi-lineage dysplasia, and in the tissue section in the bone marrow biopsy, the authors found a large cluster of PDC proliferation, and they went ahead and did the PDC-specific markers and found TCL1 and CD303 expression, proving that they were these are all plasmacytoid dendritic cells which were present along with MDS. So this should not be termed as BPDCN, whereas this is a mature plasmacytoid dendritic cell proliferation associated with MDS.

So recently, the MD Anderson group validated a 10-color flow panel which could be used for initial diagnosis as well as for MRD detection in BPDCN, and this is the 10-color flow panel. And during this validation, they also found the presence of positive reactive cells. As I mentioned earlier, that CD56 positivity was what was being used to differentiate BPDCN from mature neoplastic normal benign proliferations. However, they found that even the reactive plasmacytoid cells itself can show CD56 positivity in those cases. The other markers such as TdT, CD2, CD7, CD38, and CD303 can help us differentiate it from BPDCN. So BPDCN cells usually express CD7, CD2 is much less common in BPDCN compared to these reactive cells, and the CD2, CD38 expression is dimmer compared to the CD56 positive reactive cells. So whenever we are finding any lineage-specific antigens being negative in all our cells, before we label a case as acute undifferentiated leukemia, we should exclude the possibility of BPDCN by using a panel which is similar to this, if there is a possibility of availability of these particular antigens in our flow panel at whatever center we are using.

So the same French group also did a cytogenetic evaluation of these cases and they found that the cytogenetics show frequent losses in chromosomes with a normal karyotype being present in 30 to 40% of the cases, and around 40% showing two or more chromosomal abnormalities. Most of these are centered around the chromosome 12 and 13, which includes ETV6 and retinoblastoma gene, and also involving IKZF1. The literature database shows that the mutation frequency among the BPDCN cases. So the most commonly mutated gene is the TET2 gene, followed by ASXL1, SRF2, and NRAS, and PHF6. So these particular genes are also commonly mutated in AML, MDS, and other myeloid neoplasms, which possibly explains the coexistence or occurrence of BPDCN in conjunction with these particular neoplasms, as I had mentioned earlier.

So the prognosis, as I had mentioned that, so has a very aggressive clinical course with the median survival being around 10 to 20 months, and age seems to have an adverse prognostic impact. The other poor prognostic factors which are present in this particular disease is that high marrow or peripheral blood blast count, low TdT expression, positivity for CD303, low Ki-67 index, and the presence of deletions of cyclin D2-12 or mutations in DNA methylation genes. So the French group also did a multivariate analysis where they found that the presence of and treatment based on lymphoma therapy rather than leukemia therapy was associated with poorer prognosis.

So a little bit about the treatment. Since this is an upcoming entity and there's not much consensus on the kind of therapy which has to be given, the latest NCCN recommends that using a leukemia-like induction therapy should take precedence over using lymphoma induction. And recently, they have FDA has approved an immunotoxin which is specific for this disease, that is tagraxofusp, which is directed against the CD123 antigen. This particular drug is a modified version of the diphtheria toxin which is directed against the CD123 molecule. So this particular, the advantage of this particular disease, this particular agent has improved prognosis in this particular disease. So there is this is an ongoing thing, and there are more antibody agents and CAR T-cell directed therapies directed against CD123 which are under trial right now.

So just summarizing, when to diagnose this particular entity? Whenever on morphology, we find that there is some plasmacytoid morphology with the blast-like appearance of the nucleolus or just plain blast, and there is absence of any lineage-specific antigens of T, myeloid, or B cell lineage on flow, we should always put up the markers which is HLA-DR, CD4, CD56, and CD123. These particular markers should be readily available in most flow setups because they are commonly used. And so when we find that these particular four markers are present, we should confirm them by establishing PDC-specific lineage by doing TCL1, CD303, or CD304, any of these markers either by flow or on immunohistochemistry on tissue sections if perspective. So NCCN says that at least four of the six BPDCN antigens should be present. Of these six BPDCN antigens should be present for the diagnosis of BPDCN, and these six markers include CD4, CD56, CD123, TCL1, CD2 associated protein, and CD303, without the expression of other lineage-specific markers.

So what are the, just a few take-home messages. BPDCN is an aggressive disease arising from plasmacytoid dendritic cells. Most patients present with normal preserved WBC count and with thrombocytopenia. Diagnosis, skin is involved in most of the cases, and it's mostly the lesions are present on the face and the trunk. CNS involvement is frequent. So the immunophenotype is usually CD4, CD56, and CD123 positivity with the absence of lineage-associated antigens. So inclusion of PDC markers should be such as CD303 and CD304, and other ones which I mentioned should be included in flow or IHC to recognize this particular entity. And there can be cases which show expression of one or more myeloid antigens, and presence of mature plasmacytoid dendritic cell proliferation or plasmacytoid differentiation can be seen in myeloid neoplasms, and the prognosis is improving with the advent of targeted agents. Another additional point which I wanted to make is that before labeling any case as acute undifferentiated leukemia because of the absence of any lineage-specific antigens, we should exclude this particular entity. Thank you. Thank you so much for once again, beautiful presentation. Complete enjoyed listening to you. We'll give you this for five minutes during which we will just sort out that quiz which was asked, and then we come back to the Q&A session. So just share my screen. So okay, the quiz today was related to Rosai-Dorfman. You were given these nine statements and you were asked to pick up the wrong statement. So RDD is first described by this French pathologist in 1965, is a correct statement. It is commonly called sinus histiocytosis with massive lymphadenopathy, so that's a correct statement. We'll go straight to four. Patients can commonly present with bilateral massive painless cervical lymphadenopathy, is correct. Many patients present with extranodal disease, is correct. They may have associated autoimmune or malignant disease, is also correct. This immunophenotyping is also correct. The cells show emperipolesis, is also correct. And there is no standard care of treatment, there are no large series, so treatment is individualized, and all these modalities that have been mentioned here are correct. What was wrong is number three. The mean presentation is not 60 years. The mean age of presentation is very young, 20 years. So what you were supposed to answer was 3. And we have 9 correct answers. So 2nd to 9 are over here in this order: Dr. Sumit Milk, Dr. Sanjay Pai, Dr. Amit Karana, Dr. Pujita Reddy, Dr. Gary Punja, Dr. Savitray Singh, Dr. Pradeep Sharma, and Dr. Yoga. So congratulations to all of you. You've sent correct replies, and some of you for just few seconds after the first answer that we received. And the fastest finger first is Dr. Gurmeet Singh. Everybody knows Dr. Gurmeet Singh. He is Chief Consultant Hematopathologist and Hematologist at Jawaharlal Nehru Hospital and Research Center, Delhi. So congratulations to you. In fact, you are here right here with us. Send us your details of the bank, and we will send you a token of appreciation. So that's it. We now go to the Q&A session. Dr. Amrita, ask your question. You will have any actress whom you want to ask your question because we have three faculty members. Yes, sir. So first of all, I would appreciate, you know, it was a wonderful session by all the three speakers and full of knowledge, and I'm sure like all the listeners and all our students and fellows must have got really advantage by seeing all these academic lectures. So my question here is for Dr. Vishwat, and it was a very well-covered topic, BPDCN, and it is a very important topic, and I know like sometimes we are struggling with the diagnosis and we are stuck, and sometimes, you know, there is no flow sample, and it's a very difficult situation. We have only biopsy. So my question for him was like, if there is a flow sample, it is easier, still comparatively easier for us to go in a particular direction. But suppose we are depending on biopsy, so is there any use, I mean, what is the minimum set of panel markers which he will advise to go on biopsy in case a flow sample is not representative or it's not available? I would specifically ask like if like markers like S100, TdT, CD303, because it's a, you know, long queue of CD markers and they are expensive when we go for IHC and ask the patient to bill for a number of CD markers. So in that particular case, what is the minimum possible panel of markers on IHC on bone marrow biopsy? Thank you, ma'am. Thank you for the question. And so there have been studies on skin biopsies which have been done where they have said that use at least four or five markers. And the minimum markers which they have recommended, one of the groups, the New York group, was CD4, CD56, CD123, TCL1, and CD303, similar to what the NCCN had described that you, if you're doing it on flow, those should be included in your panel. Although I can understand that Tcl1 and CD303, these are rare IHCs which may not be available in most centers. So at least CD4, CD56, and CD123 should be included. And along with, you know, at least one or more markers for excluding other lineages, such as MPO to exclude myeloid lineage, and CD19 and CD3 to exclude the T cell lineages. So at least CD4, CD56, CD123, along with CD303 or TCL1 should be included in the IHC sections. On S100, because it has been correlated with clinical presentation, even though prognosis-wise, I'm not sure like if there is really a role of S100 positivity along with other markers in predicting prognosis. Yes, I'm not sure. So sure about S100, although it does not add any diagnostic or prognostic value even if you include it in your panel for this particular disease, at least because it may or may not be positive. So at least using the three or four markers which I mentioned and excluding the other lineages would yield a diagnosis on tissue sections. Right, but I think best is like if one can go for flow in the end. Yes. Okay, thank you. The targeted therapy for this which Vishwud mentioned is against CD123. So documentation of that has to be done. The drug is inexpensive, available under PAP through the company, and because of a rare disease, it also falls under rare disease health group, and you have been able to treat one patient from that recently. And yeah, there are trials also going on against CD303 detected antigens, I mean, agents also. So we have to see which one works out better. Yes, CD123 is FDA approved, so it's yes, the market, the tagraxofusp. Yes. Good morning everyone. Thank you, sir, for inviting me for this session. All the speakers were wonderful, and we had a good academic feast this Sunday. So my question is for Swati ma'am. It is more of a doubt and for my understanding that the first case was a case of ALL, patient of a three-year-old girl, and after 14, after I think 10 years or so, she had symptoms of weight loss and fatigue, which was later diagnosed as a mast cell disease. So my question is, and my queries that should it be called a therapy-related myeloid neoplasm with clonal mast cell disease, or should it be called a plain systemic mastocytosis with associated myeloid neoplasm? Because I know both the diseases are the same, but if we consider the age as she was diagnosed at three years of age, and in 2013, she was almost 10 years or something, so a therapy-related myeloid neoplasm with clonal mast cell disease.

would be a better diagnosis or a systemic mastocytosis with associated myeloid neoplasm. She had AL. Then she presented with bone pain, cytopenia, and massive skin. They did a splenectomy. Till then, she was in Mauritius, right? They did explain to me when she came to us, she has these mast cells, only mast cells. Because initially, I am looking only from the AL point of view. I can't document any other glass, right? So I could not document any other malignancy, any other, uh, neoplasm. And then when we reviewed her spleen, uh, sections which came from oranges, they were completely infiltrated. What they called congestive splenomegaly with mast cells. So for me to say anything other than mast cell leukemia, I have no, no support from any marker or flow. And to even say whether it was an AHN, I would not know because I don't know whether we have 14 years apart any age and which, you know, unless she was AL and also in the AHNs, AL is the least common. They say lymphomas, yes, but we don't know. So at best, I can just call it SM with AHN, but or mast cell leukemia, because when I showed you the review of the smear, they were very much there in the smear also. So I do not have anything to substantiate whether it was a therapy-induced. And you see, when they have given her 6MP with prednisone, I don't know, sir, will know is that the way to treat? Or also I don't know. So which therapy would they have given in '92 when she had her ENL? Was in '92 to develop a secondary neoplasm? I can't say. The whole case was itself so rare for me that I don't know. MR1, 6MP, I think one of the alkylating agents. And what mentions that the therapy-related neoplasms can take up to seven years or something to develop into another kind of neoplasm. Yes, so that was given in '13. '13, yeah, when they thought she relapsed. And '14, she's come to us. But what was given in '92, I don't know. Okay. As far as the chemotherapeutic drugs for AL in '92 and 2022 are unfortunate. Fortunately, they are same. We haven't evolved much on the chemotherapy front. We have immunotherapy, neutral expert, not on chemotherapy. And you're quite right to share that you have drugs like 6MP which are oncogenic. Most of the hematological malignancies develop early. The solid tumors develop late. Solid tumors can happen from median of 10 to 20 years. In hematological, from two to five years. Second, I think the largest series of 300 odd cases is from Dr. Padnani from Mayo Clinic. He was our speaker in Mumbai. And as Swati mentioned, ALL wasn't, to the best of my knowledge, wasn't at least in that series. That's about all. And from the angle of treatment, your second case which was either CMC, velour, middle story has now become the standard of care and it's available through Novartis for the last few years now. The generic is available. So the cost-efficacy has also come up. Uh, we treated one patient with interferon quite some time back, and that's one of the drugs. And, uh, cladribine is another purine analog which is effective. And your patient was treated by CMC AML protocol, you mentioned using. Yes, so they give cyto-reduction first before microscopy. Okay. Dr. Rupam.

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Everyone, and thank you very much for giving me this opportunity to be a discussion here. And thank you, Manipal Group, for sharing four very interesting cases and discussing in detail. Uh, I just want to ask Dr. Roshni, uh, if that patient of time which you discussed, do you have any follow-up of this deaf patient? Whether, because that patient also in addition had a JAK3 mutation, so that is a risk for photo development of myeloid neoplasm in Down syndrome. So I wanted to know if any follow-up was there and if the patient subsequently developed a myeloma.

We have follow-up up till August of last year, after which the patient was doing well and completely in remission. There was no secondary leukemia, at least until August. After that, we don't have follow-up. I forgot, I wanted to mention it in the presentation also, but I forgot to mention it. But let's, uh, follow up now. Post this, I don't know. I think she'll come back again for follow-up in a few months and then let's see. Hopefully, she does well. Thank you. Thank you, Doctor.

There's one question related if you want to answer, but it's on therapeutics. What should be the ideal chemotherapeutic treatment of AML with Down syndrome? I think Stockholm takes also better than I can. Low-dose, very low-dose cytarabine is what I had read in the literature, but no practical experiences. If so, has anything else to add to this?

You're absolutely right. I mean, first of all, it's not so rare. I won't say out of the three digits discussed today, that's the commonest. If you've got a pediatric oncology center and you're working over there, then this is not an uncommon disease. And now with this data, the test being available, and it should be done. I mean, you mentioned about the cost, but I think cost, I always mentioned, cost of a diagnostic test is many times cheaper than what we do on the therapeutic front. So just a little bit of talking to the patient, it's very important to look at GATA1 in this patient. So low-dose cytarabine has been the standard of care because extremely chemo-sensitive. And from that angle, we often give now drugs which we give for old people who are not fit for AML, like venetoclax, as a city. So your final goal is that you can control the disease and transplant them. Otherwise, there is no way to cure them. So your goal is cure, and that is transplant. Hello, transplant. And way to that, bridge to that is any kind of therapy, but you have to be very conscious of the doses. With extremely, extremely, they can die of infection if you try too much. And that has been the commonest, uh, happening on the clinical fronts. You give something, they get infection, they die. So you have to be very, very careful about whatever you give. Handle them like an 80-year-old AML. And yesterday, we had to talk on ALL, and because immunotherapies have come up over there, Dr. Tici mentioned that those who have developed ALL, they give only immunotherapy. On AML, we don't have that strong immunotherapy, but for ALL, you have strong chemotherapy, immunotherapy. So they give you only immunotherapy as a bridge to transplant. So Dr. Open Sharma, that was the question to your answer to your question.

Dr. Pariman Sardar, good morning, all. Thank you, sir, for giving me the opportunity as a part of decision. An excellent talk by all the three faculties of Manipal. Sir, my question to Swati, ma'am, and it was a basic question that when you suspect the mast cell proliferations on the histopathology on trephine biopsy, any morphological clue is there to suspect the mast cell proliferations on trephine biopsy?

So, are you saying that they didn't exist on the aspect? We, uh, so I, I usually see the aspirate first, right? And biopsy, yes. To me, a mast cell will look, looks like a histiocyte, unless I do markers. And, you know, they have a, so they are a variety of mast cells, like how I was, you know, that was a googly for me. A typical mast cell that you see, you can see those granules, you can see the spindling. But the problem is when there are abnormal mast cells. So there is a diffuse infiltration. Sometimes you see, I mean, most of the time you see the monocytic cleaving, you see abundant cytoplasm, you see a halo around the cells. But, and without IHC, I don't think I can highlight a mast cell in a histiocytic lesion. So my second case, I thought because it was a child dropping hemoglobin, I thought it was an earlier histiocytic proliferation. You know, if you see that, there was nothing to highlight. But then, of course, we need to do CD25 and CD2 to pick up mast cells. And Rushi, do you have anything to say on biopsy?

So either they can be spindle. I mean, there's a variety of morphology. When they're seen spindled, when you see spindle cell morphology, that's the easiest. Otherwise, they can look like histiocytes, hairy cells. You know, it's difficult on an H&E section. And we should. Are you doing the mast cell IHC on suspected cases of MPN to see whether there is MPN proliferation associated with the mast cell proliferation or anything?

No, we don't routinely do IHCs for anything. Thank you. Thank you. But we should be doing because these clones, that is small clones of mast cells that you pick up. In one of sir's previous talks, I think when Kunal spoke on CMML, that time money said that now they've started doing in all MPN, starting started putting mast cell markers. So that if they have to pick up small subsets of one thing is I am asking that in MPN, the JAK mutation can become positive because it's myeloid neoplasm. And your serum tryptase level can also be increased because of myelomyeloplasm. So if we put the IHC in that for mast cells, it will give some small clone. Whether we are dealing with the mastocytosis associated with the non-mast cell linking hematology. Yes, in MDS and CML, it is really worthwhile putting, looking for mast cells, hidden mast cells. Yeah. Thank you. Does the tryptase level differentiate?

So tryptase level can be elevated in myeloid neoplasms also. No, sir. Does the quantitation means, does the level of tryptase eye differentiate between these kind of non-specific rise versus the one in a systemic mastocytosis? Because the levels which you require for systemic mastocytosis diagnosis will not be probably seen in MPN associated disorders without without mastocytosis. And, you know, as she pointed out, Swati, you have major criteria, minor criteria. So everything to get fulfilled and still it not being a systemic mastocytosis will be a rarity. If you're looking at the mutation, you're looking at the tryptase, you're looking at the surface markers, you know, everything put together, then probably you will not make a mistake. And you have to look into all this when you're talking about such a rare disease, you must have a complete workup. So from morphology to IHC, the flow, the KIT mutation, and the tryptase.

I, I don't know in Bombay, anybody's looking at tryptase. You got your tryptase staining done from where? Outside? So at that time in '14, my colleague, these two were not here, these two young children, Reload Roshini and Rashi. I had Richard who had come from Pittsburgh from actually Upenn. So she, they sent the slide because we were upset that we had missed cases. So we wanted to do everything to prove that. And then so we sent it out before Jay came up with CD2 and CD25. You talked about Toluidine Blue. How much it helps? It really helps. That's why I said Dr. Agarwal is going to catch me on that. Toluidine Blue is not a hematology stain. You know, it's done in histopath all the time. So we didn't put it up upfront. When, so I remember this, you know, your mistakes, you never forget. Or whether it was a mistake, I don't know. When I said it is a PML-APL is a critical alert. And by the time the technician gave me MPO and so MPO were negative, I was like surprised. And I said, okay, let's go to histopath. And histo, I don't want to say they didn't treat it as an emergency. For me, APL was an emergency. And then Toluidine Blue comes at like the end of the day when you have this morphology and you are not getting MPO positivity. I think Toluidine Blue is a good stain even in this era of all these, you know, engineers and big things. I think it's a good stain too for mast cells. No, if, and even in the next case, we got it positive.

Can I help? Of course, of course. Right, Dr. Agarwal, Singh this side. Of course, please go ahead. See, uh, Toluidine Blue is very, very helpful. So far, I have reported about, I think, seven or eight cases. All of them have been initially based on Toluidine Blue. It is extensively positive and beautiful metachromatic stain and really made out. Now, in recent last five, six years, we have been using CD117 and mast cell tryptase in addition to Toluidine Blue. I think it is the cheapest stain available and very, very useful in diagnosis of mast cell disease. Must popularize it in Mumbai. They are not doing it. Okay. Thank you. Also, I have a query. Can it be used as a substitute for, uh, tryptase on IHC, since it's like cheaper in cost and it's easily available?

See, this Toluidine Blue is easily available. And wherever frozen section is done, there they have the Toluidine Blue stain. And it is always positive in normal mast cells as well as in abnormal mast cells. The only thing is here we have big clusters of mast cells which are partly replacing the bone marrow. So in a bone marrow section, you find patches of say 100, 200, 500 cells which are, uh, Toluidine Blue metachromatically positive. See, the mast cell tryptase and CD117 and CD25 and 25 are in addition to the mast cell. Toluidine Blue positivity. Ramrita, you said substitute. Substitute, you cannot use it. It is not actually. We don't have tryptase. Yeah, I don't know. So tryptase is not even in the criteria. CD2 and CD25 are in the criteria. You have to use, right? And I think it didn't do it. Remember, it's interesting. Just mention that it's a cheap stain. Right? Yes, yes, sir. Replaces the cheap stain, not tryptase at all. Costly. Tryptase is a monoclonal antibody. And it is as expensive as CD117 or any other marker. It is not extra expensive. So then it's not a problem, probably. Okay. We have to move on. Dr. Good.

My question is directed to Dr. Swati. After your excellent presentation, I just have a query regarding the prognostic factors. So apart from the common mutation, KIT mutation, that we have, do we have any other molecular markers which can be used as a prognostic factor for this systemic mastocytosis?

First of all, congratulations. Because the person who was second fastest finger was my husband. When I go back, I'm going to catch him and say, "Bye, wasn't he faster than you?" Sir is going to give you that award. So, could be, I said that you have to, you can't do only KIT mutation. You have to use the larger panel. Because especially the SRSF2, and the other two, three mutations, RUNX1, SF3B1, they are the one which are associated with adverse prognosis. It's really adverse prognosis. So KIT, along with the other, the same myeloid mutations have to be done because it puts you into the bad prognosis. And whereas the indolent ones which also have KIT but do not have others, they can go on for years. So the problem with mastocytosis is it's like bipolar. Either the indolent and smoldering do very well, and the other three do really badly. And if you have additional mutations, they are even worse. So yes, you have to do an extended panel. Thank you. Thank you.

Dr. Vithisha, um, sir, thank you for giving me this opportunity to be in this panel. My question is directed towards Dr. Roshni. So what I wanted to ask her was that how do we distinguish between TAM and acute, and acute looking associated with Down syndrome? And does this distinguish, like, does it make a difference clinically when we are treating the patient? Because if you have a one-year-old coming with 20 blasts with the same immunophenotypic features like megakaryoblastic, do we label it as myeloid leukemia associated with Down syndrome or do we label it as TAM?

Most of the patients are seen much earlier in life. So within the first three months of life is when you would call it TAM. Whereas if a patient was coming at one year, perhaps there was the prodromal syndrome, but it was just not picked up. Because I think almost all of them will have the initial TAM phase which may or may not be picked up. That's the only problem. So at one year, you'd probably still think of it as AML-DS rather than a TAM. And then based on the clinical features, you would have to decide whether you wanted to treat or not. So if the patient's asymptomatic, then you may consider waiting and watching. But I think at one year, you'd probably think of it more as AML-DS rather than a TAM.

So Roshni, you to just drop the word "probably." It is definitive. TAM timeframe is the most important. At one year, you don't diagnose TAM. Well, if you're full-blown AML at the age of three days, then you don't diagnose AML. It's the background of Down syndrome. And with availability of GATA1 and she talked about that silent TAM, you know, where GATA1 is positive but you may not have anything. So if something somebody is diagnosed at one year, even if there was no hematological features in the under, under the age of three months, it is still okay because you could have a silent case. So TAM timeframe is the most important. Twenty percent blast, even at the age of three, four, five, six days, you will not treat them because there may be total disappearance, spontaneous disappearance, spontaneous remission. All at one year, you require treatment. There is nothing like TAM there. So timeframe is the most important. Any other question, Vidisha?

Thank you, sir. There are some questions for you in the question box. As the origin of TAM is in the liver, don't we expect bone marrow blasts to be low all the time?

No. Okay. No, they can spill over. I believe so. In some patients, you do see quite a significant number of bone marrow blasts also. But overall, literature says that it's normally less. And I think in '69, it was less than the peripheral blood. So doing a marrow doesn't make too much of a difference, at least at the TAM phase. At the AML-DS, you could do it and look for fibrosis. But probably not at the TAM.

One more question lying for you is your patient, if you got GATA1 mutation in blood or was it in the bone marrow?

Blood. Bone marrow was not done. Okay. That's another question for you.

Okay, Dr. Chitra. Thank you for this wonderful session. It has been very enjoyable. My comment for Dr. Swati, you know, you emphasize that it's important not to miss such a rare case, and one sees them very seldom. So some of these cases of systemic mastocytosis may have a predominance of GI symptoms. In that situation, would there be a role for GI biopsies?

You know, most of the systemic mastocytosis cases, they have GI symptoms. Malabsorption is number one, diarrhea, peptic ulcer disease. Now, to document whether they are due to the histamine release or those mediator release or due to mast cell infiltration in the GI. So if you see the criteria, if you have something in the marrow, and you have GI symptoms, you don't need to do a GI biopsy because you have proven it in the by the major criteria. However, if you don't have a major criteria, or if your extramedullary organ is a GI itself, they weren't. So all of most of them have GI symptoms. Now, whether all of them need biopsy is the question. Biopsy is indicated when you can't document mast cells in the skin, not in the marrow. You need a GI biopsy. You need to document whether the GI symptoms are due to mast cell infiltration or the mediator release because you have lot of acid, peptic, peptic disease because of the histamine release. And also the dictum is when you are doing a GI biopsy endoscopically, if you find normal mucosa also, you go ahead and biopsy. If you can't prove infiltrates, you don't have to see abnormal. You take multiple biopsies. So yes, GI biopsies are definitely indicated when you do not have any obvious. Because for the minor criteria, you need three minor criteria. You have to establish infiltration in some, you know, extramedullary organ. So GI biopsies are definitely see indicated. But most of the, first of all, they are so rare. When you see them, they are full blown in the marrow. But in pediatrics, uh, pediatric cases with cutaneous, because cutaneous mastocytosis is commoner in children, even if you have proven cutaneous mastocytosis, you don't do a bone marrow. So in children who have GI symptoms when they have cutaneous mastocytosis, or GI biopsy is not, it's in adult mastocytosis very much. Thank you. Thank you. Very well addressed. Thank you, Dr. Chitra.

Dr. Tara, good afternoon. I really enjoyed all three talks. My queries for Dr. Swati. We have analyzed many of our cases of AMLs which had significant mastocytosis in the aspirate, that is 15 to 20% mast cells with a proven AML. And what we found was these patients, when they were treated with chemo, when they went into remission, the blast count reduced, but those mast cells persisted. And most majority of these cases relapsed very soon. So we were wondering whether it sort of indicates a bad prognosis. I mean, it's not really mentioned, but we were just, we published this thing. So have you have any experience with this where it's a proven AML with quite a significant mastocytosis?

So, um, Dr. The slide that I put up for the Austrian group, they believe firmly about this myelomastocytic leukemia, which I have never seen. Your cases qualify in those which have myeloid blasts and mast cells. Yes. So whether they were true AMLs or they are these myelomastocytic leukemias, we need to review them because they were proven with flow. We didn't have any molecular studies at that time when we, uh, when we analyzed these cases. But definite, uh, whether the mast cell was neoplastic or happened to be there, I'm not sure. But significant 15 to 20 always, I have never seen actually. After we, after the first case, when I started hunting for mast cells, because I don't work in an oncology setup here in a general hospital, I saw more mast cells coming from the cases of rheumatology, normal non-oncology cases. I see more mast cells coming from say, maybe nephro cases or mostly dermatology. But since I specifically started looking in those days, I have never found. So it's unusual. You have interesting cases that you have found 15 to 20 mast cells. I mean, it was over about a 15-year period, but I think we published around 12 of them. And I just wanted to know, I mean, all of them seem to recur. I mean, they kept relapsing. So that we thought was interesting. But you didn't establish clonality. That would be the main thing. If you ex, you have to establish neoplastic and clonal. Then they will qualify. Otherwise, it's some activation syndrome, you know, they could have something else. So now that you know, I had two even that time, but I didn't put CD2 because I didn't think of CD2. Yeah. But now that everybody has CD2, always started, maybe you can start putting that in. I just want to add one thing that, um, you know, the mast cells could be sorted using CD107 and mutation testing could be done on those to prove, you know, those specific mast cells whatever they were in your cases were truly neoplastic or not. One of the cases, CD117 was positive. But I mean, no, I'm saying you can sort them out using CD107. Use, you know, do an NGS or a molecular genetics panel on the sorted mast cells. And AFL is a disease where relapses are occurring in 80 to 90% of patients. And most of the patients can't even take the full treatment. So relapses are extremely common even otherwise. So I don't think till today we have at least a published literature that mast cell is a prognostic marker in AML. And now it's all going molecular. So I don't think you will ever have that kind of information. Only I have a question to Swati. How do you diagnose that entity which you talked about, myeloid mastocytic leukemia, which Peter Valent and Horny's group have a diagnosis?

So they said that they must have myeloid blasts. They do not have skin lesions at all. They have neoplastic mast cells. And they don't have KIT mutation. So exactly what do they have? Myeloid blasts, neoplastic cells, both? And neoplastic mast cells defined as by CD2 and CD25 markers? By markers? No KIT. No KIT.

All right. Thank you, sir. First of all, I a big thank you to Dr. Amit Agarwal for giving an opportunity as a discussion and all the speakers for a beautiful and impressive presentation. Ma'am, doctors, I haven't just want to discuss one of the cases which came in our hospital three years ago. Um, I just want to discuss with Swati, ma'am also. Basically, this lady was a 30-year-old medical officer, ma'am, who presented with, um, mild moderate pallor mainly with anemia. And on CBC was found to have a TLC of 15,000 with eosinophils of 50, 56, 757 percent, ma'am. So, we got the bone marrow done in, um, like you said, initially the bone marrow aspirate came to us before the biopsy. So in the aspirate, we had a more than 25, 30% of the mast cells, both normal and abnormal kind of a population of cells. And further, when I say came, we got the Toluidine Blue also, which confirmed that. And CD117. So subsequently, we went for CD25, which unfortunately didn't, uh, came up. So we thought it must be a non-neoplastic kind of a feature. And, uh, we went for the PDGFRA and PDGFRB also besides the BCR-ABL, just want to rule out the MPN category also. So they all came to be negative. Tryptase was very high, 22 nanogram per ml. And KIT mutation was negative. So now, we gave the hydroxyurea therapy initially, 15, 1500, and subsequently reduced the dosage and started with the PEG interferon, which continued for one and a half years. Subsequently, it has been stopped since last one and a half years. Now, this medical officer, basically, is just her other, otherwise her all other parameters have resolved, like LDH is no fuel, so TLC is, you know, gradually reducing, except for the anemia part. And we did the, uh, extensive rheumatology and other inflammatory biomarkers also workup also, but they also came negative. Um, do you think we are missing something or we can do like this in any lecture? Give authority. We'll give do the CD30 also now. Though it has, uh, it has come in the, this is the manual category now because no, it is proposed. Remember the last slide is shorter proposal. They have said that CD30 can be proposed. Your description of the case looks just like that FIP1L1-PDGFRA mutation case with eosinophilia with mast cells. But you say it was negative. So mast cells are associated with other MPNs as well. But I really can't say when you have no, also what I said, more than 90% of these cases should have KIT mutation. The question is how are we doing it? Because there are many reasons why we are getting. They are not really negative exactly because we are not doing the extensive panel. Most of them are doing 816 only, ma'am. So that that is another reason because by description, it is definitely falling into that category. But you have nothing to prove that it is so ideal. So my ex, yeah, I said, this discussion should be more of sharing experience because if you ask me, our experiences of very straight cases, we don't have. And the ones we are seeing are the bad ones. So you, at least you have a good follow-up of your case. Sorry, Dr. Ankur. I was thinking like, was there any positive control which helped you in deciding whether it's a false negative result?

False negative of, ma'am, of what? Like you said it was negative for KIT on IHC? Yes, from CD25, ma'am. Otherwise, CD117 was positive, ma'am. And actually, I got it done, um, besides our own hospital, I took the help of AIIMS, Delhi also, Dr. Pati, sir. So they are also mother, no mother significant kind of this thing. There were there some kind of a population were there, but not significantly raised to like CD117 population was there. So the, therefore, basically we came to the conclusion that there are some kind of abnormal mast cells also there. Uncle, whether this may be, initially the eosinophilia associated with the mast cell hyperplasia is there. And that's why you are not getting the KIT mutation as well as CD2 and CD25? Yes, sir. That's right. Actually, we went for PDGFRA also and, uh, also sir, FGFR1 also, sir. But, and funny JAK3 mutation also, but none of the thing gave up and gave us any indication, sir. I strongly believe, you know, a positive control should also be there always. Sometimes it's not. Yes, you can mislead in making diagnosis. Um, very true, very true. Usually, the reputed institutions always take care. But I have noted at some points, it came positive in a, you know, when we did it, and it was negative from outside. Ma'am, I mean, I don't know whether what you got was actually in our institution also, we, uh, do the positive control of each and every IHC, ma'am. If it is not there, we do the external control. So now, whether after the all the extensive workup for the inflammatory and all the patients, still whether any role of the repeat doing bone marrow biopsy and along with this ancillary technique to diagnose now a point because I think maybe that clone maybe the develops now. So we get ideas, right? Now, mother, till yesterday, I talked to the patient also. So she was, she's really asymptomatic except that her hemoglobin is between 9 to 10. The rest, her, this PNH, green part has gone away. I don't know, maybe the effect of the hydroxyurea or a PEG interferon. But I really don't know about what is basically the reason. But her hemoglobin is persistently rose. And she belongs to a region of this, this thalassemia also. So we bugged up for the, uh, this thing HPLC also, but didn't get any results. So, okay.

Dr. Sanjiv Gupta, thank you, sir, for the invitation to be part of this discussion today. And congratulations to all the speakers for their excellent, uh, cases. So my question is directed to Dr. Roshni. If she is there, in her case, GATA1 mutation was detected on NGS. So would you like to comment on the role of Sanger sequencing if the blasts are more than 20? Because most of these mutations are frameshift mutations. Or would you recommend NGS in all the cases?

From what I know, NGS is preferred over Sanger sequencing. Sanjeev, you may miss a few patients. You should. You have any additional input? I mean, it depends on what is available. Sequencing is available, that is also able to pick up. But because of the low sensitivity of Sanger sequencing, we name as some cases. Because that, as you mentioned, that there may be some unknown mutations which may cause truncation of the protein. So HPLC also has been done in some in the literature. However, since we are able to detect other, you know, gene mutations in an NGS panel, I would say NGS would be preferred over Sanger sequencing. Yeah. Because the only word is the bioinformatic pipeline has to be tuned to pick up these frameshift mutations. So although it is more sensitive, but there is a limitation in these kind of mutations. NGS. Thank you. Thank you.

Dr. Venkatesh, uh, thanks, sir. Professor Agarwal. And thanks to Atiman for giving those lovely two cases. Now we know that mastocytosis is there, and we'll be looking for it, and we'll diagnose more and more such cases in future and add to your data. Uh, regarding TAM case, I second Professor Start's that in the last one year, we have also seen two cases of TAM. And many times, it's, we, we tell the clinicians that this child is likely to have TAM and look for Down syndrome. Because in one of the unit, it was a two-year-old child, but come to it, come to us with history of intestinal obstruction. The case was referred from pediatric surgery. And we found the counts were high. And most of these cases, they also presented congenital anomalies. And they don't survive for long. They don't die of TAM, but they, we lose them because of the other congenital anomalies. So the other case had responded in two months of time. The other case had a spontaneous remission. And my question to Dr. Roshni is, how much time this child took for the spontaneous remission?

Do I think it was a couple of weeks? She took to respond. Yeah, I'm not sure exactly how many weeks she took to respond, but it wasn't too long. She went quite quickly into remission and then proceeded to relapse again. Generally, we say they remit by three months. Our gates took June. That's what the COG group had said that anything between a couple of days to 126 days is somewhere in that group. You're looking at it. So it can take a little longer also. That's it. Thanks.

Dr. Which is also so there was there's a recent paper in blood which says that flow can be used to detect that truncated GATA1s. You can detect using, you know, an intracellular stain detected against the GATA1s protein, which could be, I guess, in the future, could be used as a surrogate for, you know, mutation testing. So it's called as iGATA1s. You can use that intracellular, you know, GATA1 as protein can be used on flow on the blasts. The particular TAM blasts which you are getting. Only other point, Dr. Bank attention, is your patient was two years old. You mentioned two days. Two days. Yes. Sorry.

Good afternoon, everyone. The excellent discussion was going on. I was just a patient listener to all the points. Now I would like to make one point. It's not a question, it's just a comment that, you know, there are there are neoplasias which often manifest extra medullary sites. Two of the topics are covered in today's series of the talks. I often find that these cells, mast cells, the dendritic cells, they are often difficult to spot in the milieu of bone marrow. Often the IHC is difficult to perform on the bone marrow. Plus you add the background which, you know, gets stained with all the markers we use. I often believe, and here in Vadodara, you know, I make it a point that they always take a biopsy of the skin lesion if at all it is there in a suspected case of neoplasm. A skin lesion is much more easier to biopsy. So along with the bone marrow, when they do the skin biopsy, these cells are very easy to spot. It's easier to run the IHC markers on them because they are not decalcified tissues. It's not bones. So these sections are also crisp. Sometimes even we ask for the liver biopsy, which often is declined because of the platelet status and all. But skin biopsy, I think, is an easier thing to do. And I think we are in a position to order that biopsy to the treating clinician. And I think that that makes the things much easier. And that is what I believe. Any any take on this? Dr. Agarwal, you are a clinical hematologist, pathologist, asking for a biopsy, ordering the biopsy in such cases?

Sir, I have seen that to being helpful in lymphomas, in SHML, in this kind of situation. A rare situation of mastocytosis is where you are suspicious on bone marrow. So how do you take it? I agree with you that the number of cases of lymphomas that have been missed in the bone marrow biopsies from large number of histopathologists is phenomenal. I mean, yes. So if I may say, Dr. Other.

Small comment regarding what he said is that, uh, in a bone marrow to find biopsy is associated with increased radically. So we always almost always get a dry tap. But in a trephine biopsy, it is very easy to look for mast cells by doing both CD117 as well as mast cell tryptase. And those are brought out beautifully even in cases of AML. Dr. Was saying something in cases of AML, if we are doing CD34, CD117 on trephine biopsies, you are able to see good number of mast cells in almost every case. However, in very few cases, you may find clusters of mast cells. And that is what probably it's a little bit of mast cells are increased. Any correlation with prognosis? I am not aware of. There is another question over here about Alcian Blue. Now, Alcian Blue is not a metachromatic stain. So it cannot be a substitute for Toluidine Blue. Toluidine Blue is a beautiful stain and it gives a pinkish metachromatic staining of the mast cells. Thank you, Doctor. Thank you so much.

So coming back to Udin, I agree with you that unless you have guys like Tejinder Singh in Delhi, and we have a center in Mumbai by Dr. Jaya Mehta, the number of patients of lymphomas that are missed in the bone marrow are phenomenal. I mean, you have to be an astute hematopathologist looking at it because we depend on that. Sometimes there is no other tissue available at all. So we highly suspect that the bone marrow will be involved. And if it is missed by the hematopathologist, then the diagnosis is delayed by months together with totally changing the outcome and sometimes even death. If I am not mistaken, as far as systemic mastocytosis is concerned, and Swati is there, the major criteria is only for the bone marrow, right? It has to be either bone marrow or the extramedullary organs. And in children, then you have cutaneous mastocytosis, and you establish, you don't even have to do a marrow. So systemic mastocytosis, so yes, GI is out. Systemic mastocytosis is the major criteria. Yeah, is multifocal clustered spindle in the marrow. Cytosis, extramedullary organs. And the minor, that is minor over there, that you have to prove it in some other. That is why I was talking about the GI biopsies which are asked. So to have that three minor, then you have to have in an extramedullary organ. The major is bone marrow. Yeah.

Can I add one point here regarding the cutaneous lesion? Sanjeev, yeah. Yes. So it is not about mast cells, but talking about the cutaneous lesion, I remember one case which was BPDCN, and he had only an elbow swelling, and there was no involvement of bone marrow and peripheral blood. So we did the FNAC, took the material, and ran it for flow, and that is how we could diagnose that case. So even in BPDCN, the cutaneous lesion, because it is also a hematodermic neoplasm, so it helps in some cases. So that was our case. Thank you. Sure.

I don't see any other raised hand. Is there anybody else interested in asking anything? So I have a question. This is like, yeah, thank you, sir. Sir, it's like, in cases of TAM, like we just two days back, we had a three-day-old baby, and again, there is more than, you know, it's full of blasts. And we thought of, it could be a TAM or maybe like a needs a hematological follow-up, and then we will decide later. So I was like wondering, as a clinician, how frequently you would like to call the patient for this workup and to keep an eye on the blast count and on peripheral blood? Marrow was not done. It's just a three-day-old baby. So one marrow is not required too.

The follow-up is absolutely a must. Because when you diagnose TAM, you know that some of them will develop acute leukemia and require treatment. So to demonstrate a spontaneous regression, the only way is follow-up. So how frequently we should call the patient? Well, if it is easier, I like to call these patients every week. If it's not easy, patient is coming from outside, we call them once in a month or SOS whenever there is any clinical problem. Because usually, as was mentioned, it regresses within three months. So even if you call monthly, they're just going to come three times to you. It's not very difficult, really. It's such a major thing when you diagnose TAM, reversibility has to be shown, spontaneous possibility without you doing anything. So going back to that criteria that you said, the WHO says bone marrow and or extramedullary organs for the mast cell involvement for major. But those are more very rare. And they generally are those mast cells. So that systemic mastocytosis which you see, the literature review and all, they're all all those are documented in the marrow. But by the criteria, it can be other than marrow. Sir, is there any value of monitoring the GATA1 mutation clone in a diagnosed case of TAM?

GATA1 is important to put them in this category of TAM. But monitoring it is not important. Because not everybody is going to develop AML. GATA1 will remain. Once you talk at a somatic, this is a somatic mutation. Once that mutation has taken place, it may remain. But only about, if I'm right, about 20% develop leukemia. GATA1 itself will not be a diagnostic feature that they are going to develop leukemia. GATA1 is for 10, and then certain percentage like 20% will develop acute. And that can happen after months and years. So that drug that you had mentioned for that CD123? Yeah. So 123 is an aberrant marker now, and neoplastic. So the ones which have no pigmentation or you, or they fail mitosaurus trying that CD123 and mast cell looking at that drug that you showed and you would visualize because 123 is not seen in normal mast cells. But it's an aberrant marker on neoplastic mast cells. So it is read recently. It is a trial. Look for it. So that business was for the BPDCN. Yes. Yes. So that can be used for mast cell leukemia as well because 123 is an aberrant marker now recently being described for neoplastic mast cells. Yeah. But I suppose you're not talking about treatment, right? No, I'm just saying that we discussed that. So it was mentioned in mast cell leukemia as well. Only thing I couldn't pronounce it, so I didn't mention it. Yeah. But you know, presence of a marker doesn't mean that the treatment is going to work. Yeah, like your CD33 and you've got Gemtuzumab. Does that mean that outside APML and you're going to use that drug? There was some study I read that.

A few questions lying in the question boxes. One is, what is the cost of serum tryptase? Because he paid 12,000 rupees. So Dr. Amit Purana, you paid quite less amount. We are paying even more than that. And what is the turnaround time? So turnaround time is about three to four weeks. There is a question to Dr. Swati. Is it of any help to see increased mast cells in trephine and go ahead with further workup? We didn't discuss today at all about the other non-neoplastic causes or release patients or symptoms. But I would not pursue mast cells, you know, without any history or suspicion of elegance. So we had a 50-year-old man who had multiple fractures for no rhyme and reason. The PET scan also didn't show any disease in the marrow or in the bone. And then one of the fracture required surgical management. So whatever tissue they took out, that Dr. VPNT reported that there are mast cells. And then investigated further, and they were aberrant markers positive, and then that tryptase, tryptase was positive, and then KIT mutation, KIT mutation was positive. That came out to be just from multiple osteoporotic fracture in a younger man, 50-year-old man. It came out to be a case of systemic mastocytosis finally. So odd cases, but it was a lesson that pre-senile osteoporosis is one of the presentation of systemic mastocytosis. And then when I read the literature, full of it. Okay. So that, that's what I said. There's an entire chapter on only bone manifestations of vascular systems. And it's like an orthopedic problem. So everybody has to be aware of this. Last question lying here is about how many cases of congenital leukemia have been reported in the literature? Anyone has an exact figure?

I think it's about one in five million. But I'm not sure. Yes, a handful of cases have been reported. I don't know the figure, but it is a handful of cases.

Okay. Another question of this is it? No, that's the same question. What is lying in the chat box? Oh, that's just a thanks. All right. So I think that's that. Dr. Sanjeev, you want to ask something?

I have just a small question for Dr. Swati. Like we are talking about aberrancies in mast cells, and we rely heavily on CD25 and CD2 on flow. But we do see CD25 expression in some mast cells when we see leukemia boost inductions for MRD samples. So do you have any experience about CD25 expression in mast cells after chemotherapy in leukemias?

Because are you saying in your AML MRD, you have CD25? Is it, yeah, in some panels we did some cases with CD25 and we saw some mast cells where CD25 was there. In fact, when we search the literature, there is a paper by Sindhu Cherian where they have studied about 100 cases of non-mastocytosis, and out of that, 17% had CD25. Especially after chemotherapy. So I was just wondering if you have a similar experience.

My experience is all of two cases. And in which 25, you're using an IHC, you know, at least in the cases that we have. We have 25 on flow now. But that is why I think now that Austrian group has proposed CD30 to be used. Yeah, yeah. Because CD30 is now in cases where they don't have CD25 or CD30 expression. Yeah. But because we do only in mast cells, so we overlook the fact that it can be there in reactive mast cells also. Because if they are detected in non-mast cell disorders, CD25 is sensitive than CD2, but it is not very specific. So in fact, they suggested to raise the cutoff for CD25 positivity to 60% of the tumor cells to say that it is positive. So that increases the specificity in their study. And you are seeing this in all kinds of AMLs or some specific myeloma that we have not analyzed yet? Yeah, it is a, we have not pinpointed any specific subtype yet, but it is there. Like CD25 may be present, especially in MRD samples. So I just wanted to share that. Thanks.

Okay, so that brings us to the end of this marathon webinar, which was fantastic. Three great talks and lovely discussion following that. So right from Dr. Jinder Singh to our faculty to all the discussions and the audience for putting up questions and interest for supporting our this webinar. Thank you very much and wish you a wonderful Sunday at home. Enjoy. Bye. Thank you.

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Hey.

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