Mastocytosis and COVID19 infection and vaccination: A pediatric series

Introduction: Mastocytosis encompasses a heterogeneous group of diseases characterized by an accumulation of clonal mast cells (MC) in the skin and/or internal organs, and symptoms of MC activation. This MC activation can be elucidated by several factors, including infections or vaccination. Objective(s): We present our experience with COVID infection and vaccination in a series of 133 patients with pediatric mastocytosis. Method(s): Between January 1998 and December 2022, 133 pediatric patients have been referred to our hospital owing to clinically suspected MC disorder, mainly with mastocytosis in the skin. The final diagnoses of mastocytosis were established by the presence of typical skin lesions together with an increase of MC numbers in a biopsy from lesional skin or activating KIT mutations in lesional skin tissue. Serum baseline tryptase and total immunoglobulin E levels were measured, and patients underwent a comprehensive allergy workup to confirm atopic status and history of anaphylaxis. Regarding vaccination, REMA's (Spanish Network on Mastocytosis) protocol was followed. Result(s): 13 patients with COVID infection were identified, of which 25 (56,8%) were female and 0% had symptoms of MC activation. All of them had an asymptomatic or mild course of COVID infection. None of the patients experimented MC activation symptoms during viral illness. Regarding COVID vaccination, all patients received premedication with antihistamine 60 minutes prior vaccination. No one experimented immediate reactions and only one patient (0,75%) referred worsening of MC activation symptoms (baseline pruritus, urtication and brain fog) only after the first doses, recovering without changes in his treatment (oral cromoglycate and antihistamine) in two months. Discussion(s): Although MC have been implicated in the pathogenesis of cytokine storm in COVID19, there is no clinical evidence of SARSCoV- 2-induced MC activation, perhaps related to the fact that bone marrow MC lack angiotensin-converting enzyme 2 receptors.

Development and Utilization of an at Home Urine Exosome Biomarker Collection Test Kit to Support Urologic Telehealth

INTRODUCTION AND OBJECTIVE: COVID-19 led to paradigm shifts in telemedicine due to patient's fear of office visits and travel avoidance. With widespread cancellation of office visits and reduction of diagnostic biopsy procedures in men with elevated PSAs, the need for a non-invasive/non-DRE At Home Collection Kit for assessing risk of aggressive prostate cancer and to prioritize biopsy procedures became apparent. We adapted the existing ExoDx Prostate (EPI) office liquid biomarker kit into an At Home Collection Kit physician/ patient shared decisions for prostate biopsy. METHOD(S): A 2-stage program for an At Home Collection Testing Kit program for the ExoDx Test was initiated in April 2020 at the onset of the COVID-19 pandemic. The Phase 1 Pilot study (100 patients, 6 sites) was completed in June 2020. Based on the findings in the pilot, the program was streamlined based on feedback from physicians, patients, and office mangers before making it available in Phase 2 to all urologists in the US. The utilization of the At Home Collection Kits have been measured. RESULT(S): Extensive feedback from the pilot program led to improvements and streamlining before the Phase 2 rollout. As of October 31st, 2022, >30% of all the ExoDx Prostate Tests are At Home Collection Kits. EPI Score distributions are identical (mean 28.4 and 29.7), (median 23.0 and 24.7) in home or clinic sample collection respectively (Figure 1). CONCLUSION(S): The COVID-19 pandemic accelerated major shifts to telehealth and increased use of At Home Testing. The ExoDx Prostate (EPI) At Home Collection Kit was successfully developed and employed to help men (>50 years old) with elevated PSAs (2-10 ng/ml) considering initial or repeat diagnostic biopsy but with pandemic-related fears of visiting offices/hospitals or wanting to avoid long distance travel from rural areas. As COVID becomes manageable and clinical practices have opened, some pandemicadopted approaches remain relevant: the ExoDxTM Prostate, (EPI) At Home Collection Kit is one such approach.

FEATURES OF IMMUNOHISTOCHEMICAL MARKERS OF STEM CELLS CD-117, CD-44 IN FEMALE PLACENTAS ON THE BACKGROUND OF CHRONIC STRESS.

Background. Placenta is a subject of interest to a wide range of scientists because it is rich in stem cells and their precursors. A stem cell is a cell that has the ability to self-repair and can differentiate into offspring (daughter cells) of one or more germ layers. In recent years, scientists have obtained new data of stem cells regenerative potential. However, only isolated publications about placental stem cells are available. Therefore, our studies about placental stem cells are important for discovery of structural and molecular mechanisms, their changes under the influence of chronic stress. Objective(s): to study the features of immunohistochemical markers of pluripotent stem cells and their morphological features. Materials and methods. We examined 80 women placentas with chronic stress in comparison with control using general histological and immunohistochemical methods in the following groups: group 1 - women placentas with physiological course of pregnancy in term 38-40 weeks, group 2 - women placentas with miscarriage, group 3 - women placentas with chronic stress due to internal irradiation (4.5 Bq/kg and more), group 4 - women placentas which had COVID-19 during pregnancy. Results. There was a significant increase of stem cell markers expression in the three study groups with a significant predominance in groups 3 and 4. It was also determined the different direction of their active factors. Conclusions. The general changes of all structures of the placental barrier are detected as a result of chronic stress due to various factors: micro detachment of the decidual membrane (significant increase in cases in the studied groups);malperfusion in the structures of the maternal placental barrier;in the placenta stem cells of the three study groups in comparison with the control were found stress markers. Thus, chronic stress due to various factors causes the same type of changes in placental structures, but they have different degrees of expression - with internal irradiation doses >= 4.8 Bq/kg, these changes are most expressive.Copyright © 2022 Authors. All rights reserved.

Diagnostico Da Evolucao De Sindrome Mielodisplasica Para Leucemia Mieloide Aguda Atraves De Biopsia Intraoral: Relato De Caso

Objetivo: O presente trabalho tem como objetivo relatar um caso de evolucao de sindrome mielodisplasica para leucemia mieloide aguda, diagnosticada atraves de biopsia de lesao intraoral de sarcoma mieloide. Material e Metodos: Trata-se de um estudo descritivo, retrospectivo, do tipo relato de caso. Os dados foram coletados atraves dos registros em prontuarios eletronico e fisico da instituicao. Resultados: O caso apresentado envolveu um paciente do sexo masculino, de 51 anos, melanoderma, matriculado na instituicao devido ao diagnostico de sindrome mielodisplasica, tratado com 19 ciclos de decitabina. Devido a um quadro de odontalgia e abscessos de repeticao o paciente foi encaminhado ao setor de Odontologia onde, durante o exame clinico, foi constatada presenca de lesao eritematosa, amolecida, friavel, de superficie lisa, base sessil, de aproximadamente 3 x 1cm na regiao distopalatina do dente 26. No exame radiografico, foi observada imagem radiopaca associada a raiz mesial do dente 26, com margens definidas. Foi realizada biopsia excisional da lesao em regiao de palato com carater de urgencia e as hipoteses diagnosticas iniciais foram de granuloma piogenico e cisto radicular. O laudo histopatologico foi compativel com quadro de sarcoma mieloide comprometendo mucosa, com imuno-histoquimica positiva para CD34 e CD117 e negativa para CD163 e TdT. Diante do diagnostico previo de sindrome mielodisplasica, associado ao diagnostico de sarcoma mieloide oral, a equipe medica constatou a transformacao da doenca inicial em leucemia mieloide aguda. Foi definido novo protocolo terapeutico e realizado um ciclo de citorreducao com Citarabina (ara-C) subcutaneo e resgate com hidroxiureia + ara-c (HYDAC) + idarrubicina. Durante este ciclo, o paciente foi diagnosticado com Covid-19, evoluindo para obito, nao concluindo portanto, o protocolo terapeutico proposto. Discussao: O diagnostico de sarcoma mieloide oral permitiu que a equipe multiprofissional detectasse a transformacao da doenca primaria em leucemia mieloide aguda, resultando na reformulacao do tratamento e conduta do caso. O prognostico da leucemia mieloide aguda secundaria a sindrome mielodisplasica e pior quando comparado as primarias, bem como, apresenta baixas taxas de remissao apos quimioterapia intensiva e a sobrevida global mediana de 9-12 meses. O diagnostico dessas lesoes por parte do cirurgiao-dentista e considerado um desafio, visto que sao raras em cavidade oral e as apresentacoes clinicas costumam ser variadas e inespecificas. Conclusao: O presente relato de caso evidencia a importancia de uma avaliacao clinica minuciosa e do diagnostico preciso e completo de lesoes orais, que podem implicar diretamente na definicao da doenca de base, tratamento e prognostico do paciente. Alem disso, ratifica a importancia do cirurgiao-dentista na pratica clinica e na atuacao profissional de forma interdisciplinar e integrada com a equipe de saude. Copyright © 2022

Remissao Espontanea De Leucemia Mieloide Aguda Apos Infeccao Grave Por Covid-19 E Choque Septico: Relato De Caso

Introducao: A remissao espontanea de leucemia mieloide aguda e um evento muito raro, e suas descricoes na literatura medica geralmente envolvem um evento infeccioso grave. Mais recentemente, a COVID-19 tambem foi descrita como um dos fatores associados a tal fenomeno, e a hipotese mais aceita para o mesmo e de que ha uma hiperativacao imune que tambem apresenta efeito antitumoral. Relato de caso: Paciente feminina, de 54 anos, foi admitida no Hospital Brigadeiro com historia de alteracao de nivel de consciencia e lesao em pododactilo direito que motivou a procura do pronto socorro. No hemograma da admissao, apresentava hemoglobina 8,2 g/dL VCM 87, plaquetometria 11.000/mm3, leucocitos 99560 com 88% de celulas de medio a grande tamanho, alta relacao nucleo citoplasma nucleolos evidentes e presenca frequente de bastonete de Auer. A imunofenotipagem de sangue periferico,22,4% destas celulas revelaram marcacao CD13, CD15++, CD33++/+++, CD34 par, CD38+++, CD64+, CD71+, CD117, CD123+, HLA-DR, MPO par, alem de 18,6% de monocitos maduros, com diagnostico de leucemia mieloide aguda. Foi colhido teste rapido para COVID-19 antes da internacao, com resultado positivo. Nos 3 dias que se seguiram, a paciente evoluiu com desconforto respiratorio, taquipneia, e a tomografia de torax revelou ainda broncopneumonia sobreposta a COVID-19, o que resultou em intubacao orotraqueal por insuficiencia respiratoria. Durante a intubacao, a paciente evoluiu com parada cardiorrespiratoria em atividade eletrica sem pulso, com retorno a circulacao espontanea apos 10 minutos. Dada a gravidade do quadro, foi optado por nao iniciar inducao quimioterapica. Ao longo da internacao em UTI, a paciente fez uso de piperacilina tazobactam com vancomicina nos primeiro 7 dias de internacao, teve Pseudomonas aeruginosa multissensivel isolada de aspirado traqueal, e recebeu meropenem e teicoplanina por novo evento de choque septico, por 7 dias. Sua internacao em UTI durou 41 dias, contudo, ao longo dos primeiros 30 dias de internacao houve clareamento de blastos em sangue periferico, e parametros como hemoglobina e plaquetometria tambem evoluiram com melhora. Ao final do 42degree dia, quando recebeu alta da UTI para a enfermaria, foi feita avaliacao medular, quel nao revelou blastos com as caracteristicas imunofenotipicas previamente descritas. A paciente se encontra atualmente internada e com remissao citomorfologica e sem doenca residual minima pela imunofenotipagem. Nao foi administrada qualquer terapia antineoplasica, somente hidroxiureia para fins de citorreducao, nos primeiros 7 dias do quadro. Discussao: O caso evidencia uma rara e ainda nao compreendida remissao de leucemia mieloide aguda apos infeccao por COVID-19 e complicacoes infecciosas bacterianas graves. Casos semelhantes ja foram descritos em sepse grave e COVID-19 isoladamente, mas a evolucao natural do quadro envolve a recaida da doenca apos alguns meses do quadro agudo. A terapia antineoplasica, por sua vez, e indispensavel para a remissao sustentada. A explicacao mais aceita para o fenomeno e de que um mecanismo imunologico ativado e producao excessiva de citocinas pro-inflamatorias, sobretudo na COVID-19, destruiria as celulas neoplasicas, que poderiam inclusive ser infectadas pelo virus. Conclusao: Apesar de o mecanismo exato da remissao ser desconhecido, o caso apresentado reforca o papel da imunidade do individuo no tratamento antineoplasico e justifica a imersao em mais estudos envolvendo imunoterapia para a leucemia mieloide aguda. Copyright © 2022

EFFICIENCY OF A NEBULIZER FILTER KIT TO PREVENT ENVIRONMENTAL CONTAMINATION DURING NEBULIZER THERAPY

SESSION TITLE: Respiratory Care: Oxygen, Rehabilitation, and Inhalers SESSION TYPE: Original Investigation Posters PRESENTED ON: 10/18/2022 01:30 pm - 02:30 pm PURPOSE: The SARS-CoV-2 pandemic has highlighted the need to avoid environmental contamination with aerosols. To aid in this, the addition of a filter kit is intended to capture any exhaled aerosol. To determine the aerosol amounts emitted to the environment during nebulizer therapy several nebulizers were evaluated to test the efficiency of the nebulizer filter system. METHODS: The MaxiNeb® Duo, Circulaire® II and AeroEclipse® II BAN™ Nebulizer were operated at 50PSIG with their optional filter kits (n=5). Each device was evaluated with 2.5mg/3.0mL fill of albuterol and connected to a simulator mimicking adult tidal breathing. In addition to inspiratory and expiratory filters, the nebulizer was placed under an extraction system to capture any aerosol emitted through leakages or exhalation. Albuterol assay was undertaken by HPLC-UV spectrophotometry. RESULTS: The mass of albuterol captured from the extraction system with the MaxiNeb® Duo, Circulaire® II and AeroEclipse® II BAN™ Nebulizer was found to be 0.5±0.2, 1.5±0.6 and 0.0±0.0% of the initial dose respectively. CONCLUSIONS: The BAN™ Nebulizer without filter kit has previously reported environmental losses of just under 3%ꝉ so it is in keeping that the addition of the filter kit eliminated all losses for this device. The other two nebulizers emitted small amounts of aerosol even when a filter kit was used. CLINICAL IMPLICATIONS: If the laboratory results for the nebulizer / filter systems which did not eliminate all environmental emissions were replicated in a clinical setting, there would likely need to be an assessment performed into the potential risk to staff and patients. ꝉ Efficiency of a Nebulizer Filter Kit to Prevent Environmental Contamination During Nebulizer Therapy – presented at European Respiratory Society Meeting 2021 DISCLOSURES: Employee relationship with Trudell Medical International Please note: 27 years by Mark Nagel, value=Salary Employee relationship with Trudell Medical International Please note: >$100000 by Jason Suggett, value=Salary

NARROWING INEQUITY GAP ON WOMEN UNDERGOING HYSTERECTOMY: ERAS HOME DELIVERY KIT

Introduction: The enhanced recovery after surgery (ERAS) pathway comprises a series of evidence-based interventions accelerating recovery after surgery. COVID-19 disrupted perioperative processes, and vulnerable populations were at exceptionally high risk. Objective: To facilitate and improve adherence to preoperative ERAS pathways, preoperative chlorhexidine (CHG) and prenutritional drinks were mailed directly to patients (ERAS kit). We hypothesized that shipping kits direct to women undergoing gynecological surgery would increase adherence and provide more equitable care. Methods: This study is a retrospective cohort study of all adult cis-gender female patients undergoing gynecological surgery at a large tertiary hospital from October to November of 2021. Adherence and access to the pathway at the time of surgery were compared between White patients and other racial minority groups in October and November 2019, 2020, and 2021 (before COVID-19, during COVID-19, and intervention period). Patient demographics were described using frequency and percent for categorical variables and mean and standard deviation for continuous variables. SPC 3-sigma p-charts were used to evaluate changes in the utilization of pre-surgical ERAS interventions. Results: Compared to White patients, women from racial minority groups undergoing hysterectomy were less likely to adhere to ERAS pre-surgical interventions such as pre-surgery carbohydrate hydration (20.9 vs. 42.9%, P = 0.005) or use the preoperative CHG soap (60.4% vs. 77.6%, P = 0185). From October 1st to November 30th of 2021, a total of 127 patients that had a hysterectomy received an ERAS pre-surgery kit at home. White patients had a 91.9% adherence to pre-surgical nutrition, while other racial minority groups had 96.4% adherence (P = 0.713). During the study period, White patients had 98.0% adherence to the CHG portion of the pathway, and other racial minorities groups had 96.3% (P = 0.188). Conclusions: t baseline, non-White patients undergoing hysterectomy were less likely to adhere to ERAS pre-surgical interventions such as pre-surgery carbohydrate hydration and CHG use. Delivering ERAS pre-surgical kits directly to the patients' homes is associated with large increases in utilization of the ERAS pathway among both White patients and patients of color.

SYMPTOM STATUS AND POSTAL KIT RETURN RATES OF INDIVIDUALS ACCESSING AN ONLINE SEXUALLY TRANSMITTED INFECTION (STI) TESTING SERVICE

Background Online sexual health services (e-services) are being increasingly used for STI testing. Reduced sexual health clinic capacity due to the Covid-19 pandemic prompted our regional e-service to widen eligibility and enable individuals with mild symptoms to access our service. Prior to placing a kit order, information was shown to all symptomatic service users, advising them of the benefits of attending a sexual health clinic and/or to attend a clinic if symptoms persist/worsen. Method A retrospective analysis was performed of service users that ordered a testing kit between March 2020, and December 2021. Kit return rates were compared according to symptom status. Results Of 888,619 kit orders 163,023 (18.3%) came from symptomatic users. The most common symptom reported was vaginal discharge (41.1%), followed by dysuria/frequency (18.4%) and itching (13.8%). The kit return rate among asymptomatic service users was significantly higher (79.3%) compared to symptomatic users (74.3%) (X2 6.04. p =0.01401). The highest return rates were seen among individuals with itching (75.8%), dysuria/frequency (75.7%) and vaginal discharge (74.6%). The lowest return rate was seen by individuals with genital lumps (71.2%). Discussion Significantly fewer kits were returned by symptomatic e-service users compared to asymptomatics. It is plausible the e-service advisory information deterred some users from returning kits, encouraging them to instead test at a clinic. However, many users opted to complete testing online or perhaps did so because of difficulty accessing a sexual health clinic. Further work is needed to explore the preferred modality of testing provider and the suitability of online services for symptomatic individuals.

BEDSIDE TEST TO RULE OUT AN ECTOPIC PREGNANCY USING A MODIFIED FDA APPROVED KIT

OBJECTIVE: To evaluate the accuracy of a modified bedside test in ruling out an ectopic pregnancy. The test is based on a lateral flow immunoassay for alpha-fetoprotein (AFP). It has been shown that a high AFP level in vaginal blood indicates the passage of fetal tissue, suggestive of a miscarriage [1].We hypothesized that high AFP levels in sampled intrauterine tissue, assuming non-heterotopic pregnancy, rules out the presence of an ectopic pregnancy. MATERIALS AND METHODS: This is a prospective cohort study. The study included pregnant women undergoing a dilation and curettage (D&C) for pregnancy loss or termination, women with pregnancy loss or an ectopic pregnancy presenting with vaginal bleeding, and non-pregnant women with vaginal bleeding. Vaginal blood was collected on gauzes, sanitary pads, and cotton swabs. Samples were then tested for AFP levels using a commercial kit (ROMplus, Laborie, USA) originally designed to detect leakage of amniotic fluid. This kit contains a lateral flow immunoassay strip capable of detecting the presence of AFP. Positive samples for AFP were retested at a later date (after 3 to 20 days) to ascertain the stability of AFP and reliability of the test. Official sonograms, pregnancy tests, and final pathology results were obtained to confirm pregnancy status as well as the presence or absence of fetal tissue in the vaginal blood. A sensitivity and specificity analysis was performed against these final results to validate the accuracy of the test strip in ruling out an ectopic pregnancy. RESULTS: A total of 30 vaginal blood samples were tested for AFP. All pregnant women who had a miscarriage or D&C had detectible AFP in their vaginal blood (n=13). On retesting the samples 3 to 20 days later, these results remained the same (positive test strip). The remaining 17 vaginal blood samples were from 4 women with ectopic pregnancies and from 13 non-pregnant women with vaginal bleeding. All 4 ectopic pregnancies had no AFP detected in the vaginal blood and only 1 out of 13 non-pregnant patient samples had AFP detected. The ROMplus test strip correctly detected AFP in all samples tested containing fetal tissue (n=13) resulting in a test sensitivity of 100%. ROMplus correctly identified the absence of AFP in 16 out of the 17 samples lacking fetal tissue, a 94% test specificity. CONCLUSIONS: ROMplus has the potential to accurately and reliably detect the presence of AFP, and hence fetal tissue, in vaginal blood samples. This could be a vital non-invasive aid in ruling out an ectopic pregnancy at the bedside (currently off-label use). Furthermore, it could limit the amount of invasive testing and visits needed in cases of pregnancies of unknown location. IMPACT STATEMENT: In light of the recent COVID-19 pandemic, a simple non-invasive bedside test to rule out an ectopic pregnancy is highly desired given its potential for reducing the number of visits, investigations performed, and personnel involved in the workup of a pregnancy of unknown location.

SARCOMA MIELOIDE NA LEUCEMIA MIELOIDE AGUDA EM REMISSÃO: RELATO DE CASO

Introdução: O sarcoma mieloide tem incidência variável. Um caso de sarcoma mieloide (SM) em paciente com leucemia mieloide aguda (LMA) em tratamento com doença residual mínima (DRM) negativa será apresentado abaixo, já que preocupa as equipes médicas e se torna desafiador. Relato de caso: Paciente feminina, 21 anos, previamente hígida, queixa de astenia e dor de garganta. Procurou atendimento com 2 dias de evolução, sendo afastada infecção por SARS-CoV-2 e liberada com antibióticos. Cinco dias após houve piora dos sintomas, com astenia e surgimento de linfonodomegalias cervicais. Retornou para atendimento e laboratoriais mostraram: Hb 8,2, Ht 25,2, VCM 93, Leucócitos 90.230, blastos 88%, neutrófilos 1%, eosinófilos 1%, linfócitos 10%, plaquetas 64.000. Internada, foi submetida a análise de medula óssea (MO) que evidenciou infiltração na totalidade por blastos;imunofenotipagem (IFT) indicou LMA com diferenciação monocítica;cariótipo com trissomia do 8. Após citorredução com hidroxiureia, foi submetida a indução com Ara-C e idarrubicina (7+3), apresentando remissão morfológica em 14 dias. Seguiu esquema de consolidação com altas doses de citarabina, com DRM negativa antes do segundo ciclo. Às vésperas da terceira consolidação chega com inúmeras lesões nodulares violáceas. Biópsia da lesão gera imunohistoquímica (IHQ) compatível com sarcoma mieloide com os marcadores: mieloperoxidase, CD68 (KP1), CD33, CD34, CD4 e CD3 positivos e CD20 negativo. Com medula e sangue periférico (SP) negativos, optado por realizar mitoxantrona, etoposide e citarabina, levando a remissão da doença. Discussão e conclusão: De incidência não estabelecida, chegando até 30% de todos os casos de LMA, o SM, conforme a OMS, carecteriza-se pela massa tumoral extramedular formada por blastos mieloides com ou sem maturação. Mas incidente a partir das LMA com diferenciação monocítica, o prognóstico é incerto já que os estudos mostram de desfechos inferiores de sobrevida e tempo livre de doença até não haver mudanças entre as variáveis quando comparados com os desfechos da LMA. O SM possui uma variedade de apresentações: SM sem evidência de leucemia em SP ou MO, SM concomitante a LMA, SM associado a síndrome mielodisplásica, mieloprolifaração ou leucemia mieloide crônica e SM com LMA em remissão. Sendo a pele o principal órgão acometido a migração das células blásticas ainda não é bem compreendida, mas se evidencia relação com a expressão de CD11b, CD56 e da proteína CXCR4 mais frequente nos blastos mielomonocíticos. O diagnóstico deve ser realizado através de biópsia da lesão seguido de IHQ que inclua os marcadores CD33, CD117, MPO, CD34, CD43, CD68 (KP1), CD3, geralmente positivos. Dessa forma pode-se realizar diagnóstico diferencial com outras lesões de pele, principalmente com neoplasia de células dendríticas plasmocitoides blásticas. No estadiamento se fazem importantes os exames de imagem, preferencialmente, PET-CT, e a análise da MO, já que esta última será o critério avaliado para realização do transplante de medula óssea. O tratamento varia de acordo com a concomitância com outras neoplasias hematológicas mas, de maneira geral, os protocolos que incluem Ara-C oferecem remissão completa das lesões de pele. Diante disso, ressalta-se a importância de realizar um bom diagnóstico de forma e conhecer a patologia de forma a tranquilizar a equipe médica em relação aos desfechos.

Mast Cells in COVID-19-Associated Lung Damage

Background: SARS-CoV-2 infection results in acute respiratory distress and multiple organ failure, but the pathogenesis of the disease is poorly understood. Recent data suggest that viral RNA may be found in mast cells, but patients with asthma who have hyperplasia of mucosal mast cells do not experience asthma exacerbations during infection and do not suffer from increased mortality due to COVID-19-associated lung damage. Mast cells from bone marrow do not express the ACE2 receptor used for SARS-CoV-2 entry into human cells, and patients with mast cell activation disorders such as systemic mastocytosis do not show mast cell activation during infection. Because activated mast cells can release potent inflammatory mediators including IL-6 and have been implicated in fibrotic lung damage, the purpose of this study was to investigate the role of mast cells in COVID-19 fatal lung disease. Design: We evaluated 19 autopsies and post-mortem biopsies performed on patients who died of COVID-19 in April and May 2020. Representative sections of lung tissue with typical histological changes of diffuse alveolar damage (DAD;both acute and organizing) were selected. Mast cells were identified by immunohistochemistry for KIT (CD117), tryptase, and chymase. Mean values for mast cells were obtained by counting 3 different areas each with the highest and lowest density of CD117/tryptasepositive cells with 40x magnification. Results: Patients in this cohort were mostly obese with systemic hypertension or diabetes and had elevated CRP and IL-6. All mast cells in COVID-19 lung autopsies were positive for tryptase and chymase, indicating a connective tissue phenotype (Figure 1). While both acute and organizing forms of non-COVID-19-related lung injury showed a 3-5-fold increase in mast cell numbers between low and high-density areas, acute COVID-19 showed a <2-fold increase. In contrast, organizing DAD in COVID-19 showed a 3-fold increase in mast cells between low and high-density areas (Figure 2). Few mast cells were co-localized with SARS-CoV-2 mRNA. Conclusions: During the early phase of DAD in SARS-CoV-2 infection, mast cells are suppressed, potentially due to an interferon surge, which is reversed during the organizing DAD phase of infection. Viral RNA is rarely present in mast cells, consistent with the reported lack of ACE2 receptors in bone marrow mast cells.

Chapter 6: Screening Technologies

Screening advanced compounds enables discovery of direct repurposing candidates, novel drug-like leads for optimization, and informative pharmacological probes. In this chapter, we describe different types of screening collections used in drug repurposing, discuss issues and considerations in preparing and executing a repurposing screen, and present examples of in vitro and in vivo repurposing assays. We further describe various data sources reporting information on de-risked compounds of different types and illustrate how data mining and chemoinformatic and chemogenomic searches can be used to access large numbers of advanced compounds and assemble collections most suitable for screening in a given disease model. We argue that a view of repurposing screening as a large-scale bet on finding candidates for clinical testing is narrow and incomplete. Rather, when thoughtfully executed, screening of re-risked compounds is informed by target pathobiology and offers a means to efficiently convert advances in the development of sophisticated non-clinical models and new insights in disease mechanisms into novel drug-like leads and candidates for development.

A new translocation partner for CBFB in acute myeloid leukemia, t(10;16)(p13;q22)

Cytogenetic abnormalities involving chromosome 16 are found in 5– 8% of acute myeloid leukemia (AML). These are typically a pericentric inversion inv(16)(p13.1q22) or a translocation, t(16;16)(p13.1;q22), involving the MYH11 and CBFB genes localized to chromosome 16p13.1 and 16q22, respectively. In addition, less common rearrangements include deletion of the long arm of chromosome 16, del(16) (q22), and cryptic insertions involving the MYH11 and the CBFB genes with otherwise normal karyotypes. In this report, we present the first AML case with a new translocation involving the CBFB gene. The more common CBFB - MYH11 fusion product resulting from the inversion and/or translocation of chromosome(s) 16 leads to an AML with monocytic and granulocytic differentiation and abnormal eosinophil component with large, purple to violet color eosinophilic granules. This entity typically corresponds to the adult AML-M4Eo in French-American- British (FAB) Classification and now called AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22);CBFB-MYH1 in the new 2017 WHO Classification. Patients may present with myeloid sarcoma at initial diagnosis or at relapse. We present a case of an 80-year-old male with a history of prostate cancer post radiotherapy who was referred for COVID-19 testing. A complete blood count with differential revealed neutropenia and a macrocytic anemia. A bone marrow biopsy and a bone marrow aspirate confirmed a diagnosis of AML with 33% blasts including myeloblasts and promonocytes. Interphase fluorescence in situ hybridization (FISH) analysis with a break-apart probe for CBFB showed an abnormal hybridization pattern consistent with rearrangement of CBFB in 66% of nuclei. Chromosome analysis revealed an abnormal karyotype with two related clones: 47,XY, t(10;16)(p13;q22),+22[4]/48,idem,+8[16]. Sequential GTG-FISH confirmed that the 3’ region of CBFB was translocated to 10p13 in the t(10;16) and the 5’ region remained on 16q. Based on the karyotype, the patient’s bone barrow exhibits clonal evolution having acquired additional chromosome abnormalities (trisomy 22 and trisomy 8). Molecular studies by next generation sequencing showed NRAS p.Gln61Lys mutation with a VAF of 11.21%. No genomic alterations were detected in KIT, KRAS or FLT3 genes. AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22) is associated with a high rate of complete remission and favorable overall survival when treated with intensive consolidation therapy. However, their prognostic advantage may be affected by additional cytogenetic abnormalities and/or other gene mutations. Specifically, trisomy 22, is a frequent abnormality additional to inv(16) detected as a secondary finding which has been associated with an improved outcome when compared to the prognosis associated with inv(16) alone. Furthermore, KIT (in 30–40%), FLT3 (in 14%), NRAS (in 45%) and KRAS (in 13%) mutations are common in this AML type. The prognostic implications of KIT mutation (especially involving exon 8) do not appear to be significantly poor prognostic compared to other AML types. On the other hand FLT3-TKD mutations and trisomy 8 are associated with a worse outcome. The patient is currently receiving Vidaza 75 mg/m2, days 1–7 of a 28 days cycle with Venetoclax mg daily of a 28-day cycle and his clinical prognosis is currently unclear. Further analysis by DNA sequencing may help to characterize the molecular nature of the fusion gene product resulting from the novel t(10;16)(p13;q22). To the best of our knowledge, this is the first reported case of an AML patient with translocation t(10;16)(p13;q22) involving the CBFB gene. Given the rarity and lack of additional information regarding the effects of this abnormality, the prognosis and survival cannot be predicted.

Antibody Response to Sars-Cov-2 Vaccination in Patients Following Allogeneic Hematopoietic Cell Transplantation

Background: Vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been approved rapidly. However, pivotal studies have been conducted in healthy volunteers, while recipients of allogeneic hematopoietic cell transplantations (allo-HCT) may have different dynamics and patterns of response to the vaccine and data in this cohort is lacking. Methods: Here, we examined longitudinal antibody (AB) titers to SARS-CoV-2 vaccination with BNT162b (Comirnaty ®) or mRNA-1273 (Moderna Covid-19 Vaccine ®) in allo-HCT recipients who had undergone allo-HCT >3months (m) ago and in healthy controls (hospital employers). Serial AB titers (prior to (T0);1m after 1 st dose (T1);1m (T2), 3m (T3), 6m (T4) post 2 nd dose) were measured with an in-house developed multiplex Antibody CORonavirus Assay (ABCORA) that measures SARS-CoV-2 IgG, IgA, and IgM reactivities against RBD (receptor binding domain), S1 (subunit 1 of the spike protein), S2 (subunit 2 of the spike protein) and N (nucleoprotein), thereby allowing to differentiate immunity after vaccination versus immunity after infection. As neutralization activity correlates well with S1 AB binding, the potency of the AB response was defined as the sum of S1 IgG, IgA and IgM reactivities (cumulative S1 (cS1)). Based on computational methods high neutralization potency was predicted above a cS1 threshold of 17. Results: We enrolled 114 allo-HCT patients (median age 57y (range 18y-74y)) between March 9th 2021 and May 31st 2021 at the University Hospital Zurich, Switzerland. Currently, AB responses at T1, T2, and T3 are available for 99, 95 and 89 patients, respectively. Patients were grouped into those (A) 3-6m post-HCT (T1: n=25 at, T2: n=23, T3: n=20);(B) 6-12m post-HCT (T1: n=13, T2: n=13, T3: n=12);and (C) >12m post-HCT (T1: n=61, T2: n=59, T3: n=57). In addition, AB responses are available for healthy controls (median age 35y (range 23y-64y)) (T1: n=75, T2: n=69, T3: n=48). There were 10 patients and 5 healthy subjects with a reported or detected SARS-CoV-2 infection. There was a statistically significant difference of cS1 AB levels between the 4 groups at T1, T2, and T3 (ANOVA p-values (p) <0.001, respectively, Fig 1) with the lowest AB response in group A (cS1 median value 0.957 at T1, 5.22 at T2, 1.90 at T3) and B (cS1 median value 0.973 at T1, 4.76 at T2, 11.9 at T3) compared to group C (cS1 median value 6.21 at T1, 199 at T2, 76.4 at T3) and healthy controls (cS1 median value 54.9 at T1, 228 at T2, 91.1 at T3). Using a multivariate linear regression analysis adjusted on age and gender, we found that patients in groups A and B had significantly lower cS1 levels than groups C and healthy subjects (p<0.001, p<0.001, p=0.034 of healthy versus groups A, B, C respectively at T2, and p<0.001, p=0.004, p=0.12 at T3), and that preinfected patients had higher cS1 levels at T2 and T3 respectively (p=0.003 and 0.006). The dynamics of the AB response were more diverse in allo-HCT recipients. In a multivariate linear regression analysis (Fig 2) assessing factors associated with humoral immune responses in allo-HCT recipients, we found consistently lower cS1 responses in patients early post-HCT (group A+B (p=0.002)) and higher cS1 levels in those who had been preinfected with SARS-CoV-2 (p=0.012). Patients under immunosuppressive treatment (IST) and those who had relapsed disease post-HCT showed significantly lower cS1 immune responses (p=0.028 and 0.005, respectively). The presence of moderate or severe chronic GVHD was not a statistically significant factor influencing AB levels. This may be explained by (i) the heterogeneity of the condition of chronic GVHD and low patient numbers;(ii) the late time point >12m post-HCT with generally higher AB levels. Consistent with other reports age >65y was also associated with lower cS1 responses (p=0.03). Conclusion: Allo-HCT recipients early post-transplant, those of older age, and those given IST displayed insufficient AB titers to the vaccine. Such knowledge is of critical importance to transplant recipients and th ir physicians to guide treatment decisions regarding re-vaccination, and social behavior during this pandemic. Monitoring AB development in all allo-HCT recipients and vulnerable patients with other immunocompromising conditions may be crucial to determine those at increased risk for infection and for the timing of booster vaccines. [Formula presented] Disclosures: Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company;University of Zurich: Patents & Royalties: CD117xCD3 TEA.