Pathology Playground: An Interactive Web-Based System for Pathology Unknowns With Immunohistochemistry and Molecular Studies

(Arch Pathol Lab Med. 2023;147:492-495;doi: 10.5858/ arpa.2021 -0516-EP) Interactive, computer-based learning has been used in numerous areas of medicine, and more often than not is more effective than traditional teaching methods.1,2 Patient simulations are even used in the United States Medical Licensing Examination to assess diagnostic decision-making skills, therapeutic intervention skills, and patient management skills.3 Computer-aided learning in pathology has been used in the forms of virtual slides linked to additional content,4 games in which correctly answering questions results in saving a patient or earning virtual prizes,5 and programs that aid the user in following an algorithmic approach to histologic diagnosis.6 Additionally, in the COVID-19 era, Web-based learning is proving more valuable in replacing standard formats.7 Working through a difficult case in pathology typically involves the use of immunohistochemistry and other special studies, including molecular diagnostics, that either confirm a suspected diagnosis or narrow the differential diagnosis and guide further tests. Ideally, a trainee would be able to see the H&E slide, choose what to order, refine the diagnosis, and perhaps order further studies, which would be closer to clinical practice. Images were obtained from in-house cases as well as online sources, primarily the University of Michigan Virtual Slide Box.8 An interface was written in HTML, PHP (PHP Hypertext Preprocessor), and JavaScript. About 50 cases have been presented in this manner in total, such as carcinoid tumor of the pancreas (with synaptophysin and chromogranin), blastic mantle cell lymphoma (with immunohistochemistry, flow cytometry, and fluorescence in situ hybridization [FISH] results), synovial sarcoma (with immunohistochemistry and cytogenetics results), hemochromatosis (with special stains and molecular genetic results confirming the patient's mutation), and histiocytic sarcoma (with immunostains).

Atypical presentation of covid-19 as acute leukemia: A case report

In the absence of respiratory system involvement, COVID-19 patients developing ARDS can clinically mimic other diseases including acute leukemia due to presence of atypical lymphocytes in peripheral blood smear and increased serum lactate dehydrogenase and serum uric acid levels. Herein, we report a case who was initially suspected to have acute leukemia based on his atypical symptoms without any respiratory system involvement and deranged laboratory parameters and finally, diagnosed with COVID-19. Our patient presented with fever, myalgia, gum bleed, and petechiae. On clinical and laboratory evaluation, he was suspected to have acute leukemia based on markedly deranged serum lactate dehydrogenase and serum uric acid and the presence of atypical cells in peripheral blood smear and bone marrow. On day 3 of hospitalization, he developed respiratory symptoms, breathing difficulty which progressed to ARDS, and subsequently, he succumbed to his illness. His real-time reverse transcriptase-polymerase chain reaction test for severe acute respiratory syndrome coronavirus-2 yielded positive results. Also, Flow cytometry and fluorescence in situ hybridization studies for leukemia workup did not show any abnormalities. Although we are reporting the findings of only a single case, we aim to enhance and contribute further to the understanding of this novel infection.Copyright © 2021 Necati Ozpinar. All right reserved.

Development of a high-sensitivity and short-duration fluorescence in situ hybridization method for viral mRNA detection in HEK 293T cells.

Coronavirus disease 2019 (COVID-19) is an extremely contagious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Early disease recognition of COVID-19 is crucial not only for prompt diagnosis and treatment of the patients, but also for effective public health surveillance and response. The reverse transcription-polymerase chain reaction (RT-PCR) is the most common method for the detection of SARS-CoV-2 viral mRNA and is regarded as the gold standard test for COVID-19. However, this test and those for antibodies (IgM and IgG) and antigens have certain limitations (e.g., by yielding false-negative and false-positive results). We have developed an RNA fluorescence in situ hybridization (FISH) method for high-sensitivity detection of SARS-CoV-2 mRNAs in HEK 293T cell cultures as a model. After transfection of HEK 293T cells with plasmids, Spike (S)/envelope (E) proteins and their mRNAs were clearly detected inside the cells. In addition, hybridization time could be reduced to 2 hours for faster detection when probe concentration was increased. Our approach might thus significantly improve the sensitivity and specificity of SARS-CoV-2 detection and be widely applied for the high-sensitivity single-molecular detection of other RNA viruses (e.g., Middle East respiratory syndrome coronavirus (MERS-CoV), Hepatitis A virus, all influenza viruses, and human immunodeficiency virus (HIV)) in various types of samples including tissue, body fluid, blood, and water. RNA FISH can also be utilized for the detection of DNA viruses (e.g., Monkeypox virus, human papillomavirus (HPV), and cytomegalovirus (CMV)) by detection of their mRNAs inside cells or body fluid.

Haematological Malignancy Presenting in an Unusual Manner

Chronic lymphocytic leukaemia is a haematological malignancy that occurs due to an increased proliferation of mature B lymphocytes. It is considered to be the most common leukaemia in adults. Hyponatremia is commonly seen in such patients. This case report is about a 75-year-old male, who presented with giddiness, followed by altered sensorium. However, the patient had no motor weakness or sensory loss. Initially, a diagnosis of posterior circulation stroke was made but Magnetic Resonance Imaging (MRI) brain did not show associated signs. The routine investigations showed highly elevated total leukocyte count and hyponatremia. The patient was worked up for malignancy and diagnosed with Chronic lymphocytic leukaemia. Oncology reference was taken and treated with tablet Ibrutinib. On discharge, the patient's mentation improved, and he is on regular follow-up.

SPECIFIC DETECTION and REPLICATION KINETICS of SARS-CoV-2 USING smRNA-FISH

Background: SARS-CoV-2 is a positive-sense single-stranded RNA virus and its replication begins after the synthesis of virally encoded polymerase complex that is required for replication and transcription of genomic RNA (gRNA) within the infected cells. Despite the global interest in the study of SARS-CoV-2, the kinetics of SARS-CoV2 RNA replication and transcription during the early phase of viral infection is poorly understood. Here, we used the single-molecule RNA fluorescence in situ hybridization (smRNA-FISH) for sensitive detection of SARS-CoV-2 at single molecule level and to determine the replication of genomic RNA (gRNA) and sub-genomic RNA (sgRNA) in the infected cells, at very early stages of infection. Methods: We designed highly specific smRNA-FISH probes targeted to gRNA and Spike gene sgRNA of SARS-CoV-2 virus, using stellaris method and optimized the method to simultaneously visualize these two RNAs at single cell and single molecule level. Because of the high sensitivity of our probes, we applied smRNA-FISH technology to detect SARS-CoV-2 positive cells from autopsy samples obtained from diseased COVID-19 patients. Furthermore, we used high-resolution and high-speed scanning microscopy to detect extent of infection in cell models of SARS-CoV-2 and in COVID-19 patient samples. Results: A time course analysis SARS-CoV-2 replication indicated that single molecules of gRNA could be detected as little as 30 min to 2 hr. post-infection. Distinct "Replication Centers" (RC) began to appear one to two hours post-infection and the sgRNAs began to migrate out of these RCs. Replication after the initial delay appeared to be rapid and gRNA and sgRNAs dispersed throughout the cell within 4-5 hours post infection forming multiple RCs. We found that our RNA-FISH correctly detected the SARS-CoV-2 positive samples from patient autopsy samples that were characterized by qRT-PCR or immunological detection methods. The signals of spike gRNA and sgRNA along with the spike proteins co-localized within the same cells of the SARS-CoV-2 infected patients within the cells of lung, kidney, and heart autopsy samples. Conclusion: We propose that the specific probes and the methodology that we have developed will be highly applicable to the study of SARS-CoV-2 replication in depth and to characterize SARS-CoV-2 infection in COVID-19 patient samples. This study may open a novel direction towards COVID-19 pathophysiology, drug screening and diagnostics.

RESTRICTED INFECTION of MACROPHAGES by SARS-CoV-2 INDUCES PROINFLAMMATORY RESPONSES

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with immune hyperactivation and high levels of proinflammatory cytokines. Extensive lung infiltration by CD169+ inflammatory monocytes and presence of activated CD169+ alveolar macrophages suggest monocyte/macrophages are key drivers of severe morbidity and mortality. In this study, we determined whether CD169 mediated ACE2-independent SARS-CoV-2 entry and restricted viral genome replication in macrophages triggers pro-inflammatory cytokine expression. Methods: Monocyte-derived macrophages (MDMs) and PMA-differentiated THP-1 macrophages engineered to constitutively express CD169, ACE2, or CD169 and ACE2 were infected with USA-WA1/2020/SARS-CoV-2 isolate with or without Remdesivir pre-treatment. To identify mechanism of innate immune activation, nucleic acid sensing pathways were selectively depleted in CD169+ macrophages. Extent of viral genomic (gRNA) and sub-genomic (sgRNA) expression and induction of pro-inflammatory cytokines was determined by qRT-PCR and single molecule RNA FISH analysis. Viral protein expression and infectious virus particle production was determined by immunofluorescence analysis and TCID50. Results: While productive virus infection (viral protein expression and infectious virus particle release) was only observed in ACE2+ macrophages, SARS-CoV-2 N or S expression and infectious virus production was not observed in CD169+ macrophages. Co-expression of ACE2 and CD169 significantly enhanced infectious virus production and spread. Interestingly, smFISH and RT-qPCR analysis revealed CD169+ cells express cytosolic negative-strand gRNA and positive strand sgRNA. Importantly, CD169-mediated SARS-CoV-2 infection of macrophages and expression of viral mRNAs led to induction of pro-inflammatory cytokines, IL-6, TNFα, and IL-1β, despite lack of viral protein expression in CD169+ macrophages. Pre-treatment with Remdesivir blocked de novo expression of viral mRNAs and induction of inflammatory cytokines in CD169-dependent infection of macrophages. Furthermore, knockdown of cytosolic RLRs (RIG-I and MDA-5) or MAVS significantly attenuated inflammatory cytokine expression in CD169+ macrophages, confirming that nucleic acid sensing of restricted cytosolic viral mRNA expression in macrophages triggers innate immune activation. Conclusion: These results suggest that restricted SARS-CoV-2 infection of CD169+ macrophages contributes to COVID-19-associated hyperinflammatory cytokine response.

Fluorescence In Situ Hybridization (FISH) Tests for Identifying Protozoan and Bacterial Pathogens in Infectious Diseases.

Diagnosing and treating many infectious diseases depends on correctly identifying the causative pathogen. Characterization of pathogen-specific nucleic acid sequences by PCR is the most sensitive and specific method available for this purpose, although it is restricted to laboratories that have the necessary infrastructure and finance. Microscopy, rapid immunochromatographic tests for antigens, and immunoassays for detecting pathogen-specific antibodies are alternative and useful diagnostic methods with different advantages and disadvantages. Detection of ribosomal RNA molecules in the cytoplasm of bacterial and protozoan pathogens by fluorescence in-situ hybridization (FISH) using sequence-specific fluorescently labelled DNA probes, is cheaper than PCR and requires minimal equipment and infrastructure. A LED light source attached to most laboratory light microscopes can be used in place of a fluorescence microscope with a UV lamp for FISH. A FISH test hybridization can be completed in 30 min at 37 °C and the whole test in less than two hours. FISH tests can therefore be rapidly performed in both well-equipped and poorly-resourced laboratories. Highly sensitive and specific FISH tests for identifying many bacterial and protozoan pathogens that cause disease in humans, livestock and pets are reviewed, with particular reference to parasites causing malaria and babesiosis, and mycobacteria responsible for tuberculosis.

Pancarditis as the Clinical Presentation of Eosinophilic Granulomatosis with Polyangiitis: A Multimodality Approach to Diagnosis

Eosinophilic pancarditis (EP) is a rare, often unrecognized condition caused by endomyocardial infiltration of eosinophil granulocytes (referred as eosinophilic myocarditis, EM) associated with pericardial involvement. EM has a variable clinical presentation, ranging from asymptomatic cases to acute cardiogenic shock requiring mechanical circulatory support (MCS) or chronic restrictive cardiomyopathy at high risk of progression to dilated cardiomyopathy (DCM). EP is associated with high in‐hospital mortality, particularly when associated to endomyocardial thrombosis, coronary arteries vasculitis or severe left ventricular systolic dysfunction. To date, there is a lack of consensus about the optimal diagnostic algorithm and clinical management of patients with biopsy‐proven EP. The differential diagnosis includes hypersensitivity myocarditis, eosinophil granulomatosis with polyangiitis (EGPA), hypereosinophilic syndrome, parasitic infections, pregnancy‐related hypereosinophilia, malignancies, drug overdose (particularly clozapine) and Omenn syndrome (OMIM 603554). To our knowledge, we report the first case of pancarditis associated to eosinophilic granulomatosis with polyangiitis (EGPA) with negative anti‐neutrophil cytoplasmic antibodies (ANCA). Treatment with steroids and azathioprine was promptly started. Six months later, the patient developed a relapse: treatment with subcutaneous mepolizumab was added on the top of standard therapy, with prompt disease activity remission. This case highlights the role of a multimodality approach for the diagnosis of cardiac involvement associated to systemic immune disorders.

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.

Molecular underpinnings of olfactory dysfunction following SARS-CoV-2 infection

Olfaction is a closely coordinated partnership between odorant flow and neuronal signaling. Disruption in our ability to detect odors, or anosmia, has emerged as a hallmark symptom of infection with SARS-CoV-2, and yet, decoding the mechanism behind this abrupt sensory deficit remains elusive. Patients with COVID-19 lack symptoms of nasal congestion and rhinorrhea present in many upper respiratory tract infections that result in a conductive reduction in an ability to perceive smells. To investigate the molecular underpinnings of SARS-CoV-2 related smell loss, we performed molecular analysis, including scRNAseq, RNA-FISH, and Hi-C on both human and syrian golden hamster olfactory epithelium. Here, we report that smell loss may be attributable to non-cell autonomous mechanisms that induce genomic compartment dysregulation and subsequent downregulation of critical signaling pathways responsible for production of olfactory receptors.

Avenue - Avelumab in the Frontline Treatment of Advanced Classic Hodgkin Lymphoma - a Window Study

AVENuE - Avelumab in the frontline treatment of advanced classic Hodgkin lymphoma - a window study Background Response adapted ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) has become a standard of care in many countries for advanced stage classic Hodgkin Lymphoma (cHL), as investigated in the RATHL study: following 2 cycles of ABVD patients with negative (Deauville 1-3) interim PET (iPET2) proceeded to 4 cycles of AVD;those with positive (Deauville 4-5) iPET2 intensified therapy to escalated bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisolone (escBEACOPP) or BEACOPP every 14 days. Overall this strategy was associated with a 3-year progression free survival (PFS) of 82.6%, and outcomes for patients with positive iPET2 were disappointing with 3y progression-free survival (PFS) of 67.5%. More intensive treatment such as upfront use of escBEACOPP has been reported to produce higher PFS (89% at 5 years), but it is unclear whether overall survival (OS) is improved. More intensive treatment is, however, associated with higher risk of toxicity. Inhibitors of programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) have established efficacy in relapsed / refractory cHL with response rates of 55-87%. In the front line setting PD-1 inhibitors have a reported complete metabolic response (CMR) rate of 18-37%. Response to PD-L1 inhibitors in the frontline setting has not been explored. Serial serum TARC (thymus and activation-regulated chemokine) is reported to be prognostic in the frontline treatment of cHL and may aid response assessment because PET interpretation with checkpoint inhibitors is often complex. In the context of PD-1 inhibition, PD-1 expression by immunohistochemistry (IHC) and 9p24.1 copy gain by fluorescence in situ hybridisation (FISH) are reported to correlate with response. Methods AVENuE is a Phase II single-arm multicentre study with sites in the UK and Australia assessing the safety and efficacy of 2 cycles (4 doses) of the PD-L1 inhibitor avelumab for untreated high-risk stage II-IV cHL prior to the iPET2 response adapted approach described above. Eligible patients must be 16-60 years, ECOG 0-1, and have adequate organ function. Patients with;compressive symptoms from lymphoma, autoimmune disorders or immunosuppressive treatment within 2 months are excluded. The primary endpoint is the centrally reviewed PET CMR rate to avelumab. Secondary endpoints are: the safety and tolerability of sequential avelumab and combination chemotherapy as assessed by CTCAE v 5.0;the iPET2 CMR rate after avelumab and 2 cycles of ABVD;PFS and OS at one year. Using a single stage A'hern design, target recruitment is 47 patients to give 90% power at a 0.05% one sided alpha to exclude an overall response rate (ORR) to 2 cycles of avelumab of < 20%;an ORR of 40% would be considered worthy of further study. Recruitment has continued during the COVID-19 pandemic. 29 patients have been enrolled. Exploratory endpoints include correlating disease response with baseline PD-1 copy number by FISH and PD-1 expression by IHC. Serial serum TARC is being explored as an aid to response assessment and changes in peripheral blood immune cell subset are being investigated as possible biomarkers of response. Trial funder: Pfizer Ltd in alliance with Merck KGaA Pfizer Ltd is providing funding as part of an Alliance between Pfizer and Merck KGaA Clinical trials.gov NCT03617666 EUDRACT No.: 2018-002227-42 Disclosures: Hawkes: Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel and accommodation expenses, Research Funding, Speakers Bureau;Regeneron: Speakers Bureau;Merck KgA: Research Funding;Astra Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau;Janssen: Speakers Bureau;Merck Sharpe Dohme: Membership on an entity's Board of Directors or advisory committees;Novartis: Membership on an entity's Board of Directors or advisory committees;Gilead: Membership on an entity's Boa d of Directors or advisory committees;Antigene: Membership on an entity's Board of Directors or advisory committees;Bristol Myers Squib/Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding;Specialised Therapeutics: Consultancy. Barrington: Bristol Myers Squibb international corporation: Research Funding;Pfizer Inc: Research Funding;Amgen Ltd: Research Funding;Takeda Speakers Bureau: Honoraria. McKay: Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees;Gilead: Honoraria, Other: Travel Support;KITE: Honoraria, Membership on an entity's Board of Directors or advisory committees;Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Support;Janssen: Honoraria, Other: Travel Support;Beigene: Honoraria, Membership on an entity's Board of Directors or advisory committees;BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Iyengar: Janssen: Other: conference support, Speakers Bureau;Abbvie: Other: conference support;Beigene: Membership on an entity's Board of Directors or advisory committees;Gilead: Membership on an entity's Board of Directors or advisory committees, Other: conference support, Speakers Bureau;Takeda: Membership on an entity's Board of Directors or advisory committees, Other: conference support, Speakers Bureau. Radford: Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau;AstraZeneca: Current holder of individual stocks in a privately-held company;ADC Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company, Honoraria, Speakers Bureau;BMS: Honoraria. Shah: Abbvie, Janssen and Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Clifton-Hadley: Bristol-Myers Squibb Pharmaceuticals Ltd.: Other: The haematology team at the CTC has received funding (which in part pays staff salary) to Sponsor and coordinate clinical trials.;Amgen: Other: The haematology team at the CTC has received funding (which in part pays staff salary) to Sponsor and coordinate clinical trials.;Celgene: Other: The haematology team at the CTC has received funding (which in part pays staff salary) to Sponsor and coordinate clinical trials.;Merck Sharp and Dohme: Other: The haematology team at the CTC has received funding (which in part pays staff salary) to Sponsor and coordinate clinical trials.;Janssen-Cilag: Other: The haematology team at the CTC has received funding (which in part pays staff salary) to Sponsor and coordinate clinical trials.;Pfizer: Other: The haematology team at the CTC has received funding (which in part pays staff salary) to Sponsor and coordinate clinical trials.;Millennium pharmaceutics inc.: Other: The haematology team at the CTC has received funding (which in part pays staff salary) to Sponsor and coordinate clinical trials. Collins: Beigene: Membership on an entity's Board of Directors or advisory committees;Novartis: Honoraria, Speakers Bureau;Pfizer: Honoraria;Celgene: Research Funding;Amgen: Research Funding;AstraZeneca: Honoraria, Research Funding;ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees;Celleron: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding;Merck Sharp & Dohme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding;Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau;Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Speakers Bureau. OffLabel Disclosure: Avelumab prior to frontline chemotherapy in advanced stage classic Hodgkin lymphoma.

Cybord-Dara in Newly Diagnosed Transplant-Eligible Multiple Myeloma: Follow up Results from the 16-Bcni-001/Ctrial-IE 16-02 Study Show High Rates of MRD Negativity at End of Treatment

The anti-CD38 monoclonal antibody Daratumumab has shown impressive activity in combination with other agents for the treatment of multiple myeloma (MM), improving progression free survival and overall survival in several phase 3 studies. We conducted a phase 1b study of intravenous Daratumumab (16 mg/kg) with weekly subcutaneous bortezomib (1.3-1.5 mg/m 2 ), cyclophosphamide (150-300 mg/m 2), and dexamethasone (40 mg) (CyBorD-DARA) as induction before autologous stem cell transplantation (ASCT), followed by CyBorD-DARA consolidation (2 cycles) and monthly DARA +/- bortezomib (in high-risk disease) maintenance for 24 months. We hypothesized that the addition of cyclophosphamide could lead to enhanced antibody dependent cellular phagocytosis (ADCP). This trial was registered at www.clinicaltrials.gov as NCT02955810. We previously reported the initial results of this study. 1. In addition to a favourable safety profile we observed promising anti-MM activity with 10 of 13 patients (77%) in whom assessment was possible achieving measurable residual disease (MRD) negativity at a sensitivity of 10 -5 by next generation sequencing (NGS) after ASCT. We now report the results at EOT, with a focus on MRD. Eligible patients were ≤70 years of age with untreated transplant-eligible MM. 18 patients were enrolled. Median age was 56.5 years (range, 32-66 years), 61% were male and 94% of patients had Eastern Cooperative Oncology Group performance status ≤1. The International Staging System stages were I, II, and III in 78%, 17%, and 6% of patients, respectively. 29% of patients had high-risk genetic features by fluorescent in situ hybridisation (FISH) or gene expression profiling, including 17p deletion in 12% and t(4;14) and t(14;16) in 6% each. On an ITT basis, the rates of very good partial remission or better (≥VGPR) after ASCT, consolidation and at end of treatment (EOT) (after completion of 24 months of DARA) were 94%, 94% and 81% respectively, and rates of complete response or better (≥CR) were 50%, 63% and 63% respectively. Measurable residual disease (MRD) assessment was possible in 13 patients after induction, ASCT and consolidation, and 10 at EOT. Sustained MRD negativity (ie. MRD negativity after ASCT, consolidation and at EOT) to a level of 10 -5 by NGS was achieved in 33% (ITT). Of 13 patients who remained on study at EOT in VGPR or better, 54% were MRD negative (MRD was unavailable in 23%). 7 patients were MRD negative after both ASCT and consolidation. Of these patients, all evaluable at EOT(6/7) remained MRD negative, with 1 patient unable to undergo MRD assessment due to the COVID-19 pandemic, but remaining in CR. Nausea and diarrhoea occurred in 89% of patients, but were mostly grade 1-2 (Grade ≥3 nausea 17%;diarrhoea 6%). Neutropenia occurred in 44% (Grade ≥3 17%), anaemia in 39% (Grade ≥3 22%), and thrombocytopenia in 33% (Grade ≥3 22%). The rate of neutropenic sepsis was 11%. Infusion-related reactions occurred in 50% (Grade ≥3 6%) and peripheral neuropathy occurred in 33% (Grade ≥3 0%) The most commonly reported serious adverse event (SAE) was sepsis in 22%. One patient developed abnormal liver function tests leading to discontinuation from the trial. CyBorD-DARA induction, consolidation and DARA-maintenance is an effective and well-tolerated IMiD free regimen in transplant-eligible patients with MM. MRD negativity at a level of > 10 -5 after ASCT and consolidation may be predictive of sustained MRD negativity at EOT. References: 1. Naicker SD, et al. Cyclophosphamide alters the tumor cell secretome to potentiate the anti-myeloma activity of daratumumab through augmentation of macrophage-mediated antibody dependent cellular phagocytosis. Oncoimmunology. 2021 Jan 25;10(1):1859263. doi: 10.1080/2162402X.2020.1859263. PMID: 33552684;PMCID: PMC7849715. 2. O'Dwyer M, et al. CyBorD-DARA is potent initial induction for MM and enhances ADCP: initial results of the 16-BCNI-001/CTRIAL-IE 16-02 study. Blood Adv. 2019 Jun 25;3(12):1815-1825. doi: 10.1182/bloodadvances.2019000010. PMID: 31201169;PMCI : PMC6595251. Disclosures: O'Dwyer: ONK Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees;Janssen: Consultancy;Bristol Myers Squibb: Research Funding. Quinn: Takeda: Honoraria. Szegezdi: ONK Therapeutics: Research Funding.

Acquired Aplastic Anemia in Immunocompetent Pediatric Patients with Asymptomatic COVID-19 Infection: A Case Series

Background: In pediatrics, acquired aplastic anemia (AA) is most commonly due to infection, particularly viruses, when a cause can be identified. Coronavirus disease 2019 (COVID-19) has affected more than 197 million people worldwide, and children typically experience a less severe disease course. COVID-19 is known to cause transient hematologic abnormalities, including leukopenia, lymphopenia, anemia and thrombocytosis or thrombocytopenia in severe cases. Objectives: Describe three cases of COVID-19 associated acquired aplastic anemia in immunocompetent pediatric patients. Design/Methods: Case series established by retrospective review of the electronic medical record. Results: Case 1: An 8-year-old Hispanic male presented with a three-week history of increased bruising and a one-week history of progressive exercise intolerance, shortness of breath, pallor and fatigue. Labs showed pancytopenia. Bone marrow aspirate and biopsy was markedly hypocellular at 5-10% consistent with aplastic anemia (Figure 1). Work-up for the etiology of his aplastic anemia was only significant for positive SARS-COV-2 antibodies and a SEC23B variant of unknown significance on a comprehensive bone marrow failure (BMF)/myelodysplastic syndrome (MDS)/leukemia panel from the Children's Hospital of Philadelphia (CHOP). He was treated with eltrombopag olamine and then proceeded to immunotherapy with cyclosporine (CsA) and horse antithymocyte globulin (ATG) when a sibling match was not identified for hematopoietic stem cell transplant (HSCT). Three months later, his peripheral blood counts have improved, and he is no longer transfusion-dependent. Repeat bone marrow aspirate and biopsy continues to show markedly hypocellularity at <5%. Case 2: A 5-year-old non-Hispanic white female presented with a two-week history of easy bruising, petechial rash, fatigue and bone pain. Labs showed pancytopenia, and bone marrow aspirate and biopsy showed marked hypocellularity at 5-10% consistent with aplastic anemia (Figure 2). Her aplastic anemia work-up was significant for positive SARS-COV-2 antibodies and subclinical RBC and WBC paroxysmal nocturnal hemoglobinuria (PNH) clones. She was started on eltrombopag olamine and then proceeded to immunotherapy with CsA and ATG when a matched sibling donor was not identified. Three months later, she continues to be severely neutropenic, anemic and thrombocytopenic requiring multiple transfusions. Repeat bone marrow aspirate and biopsy showed variable cellularity with some areas 10-20% and others 70% with an overall cellularity of 50%. Case 3: An 8-year-old non-Hispanic white female presented with a 10-day history of fatigue, bilateral leg pain and pallor. Labs showed pancytopenia, elevated inflammatory markers and elevated hemoglobin F. Bone marrow aspirate and biopsy demonstrated mild-moderate hypocellularity at 40-50%, left-shifted myelopoiesis and dyspoiesis in the erythroid and megakaryocytic cell lines (Figure 3). MDS and acute lymphoblastic leukemia (ALL) fluorescence in situ hybridization (FISH) panels were negative. Additional work-up revealed positive SARS-COV-2 antibodies. Her pancytopenia resolved within two weeks of her initial hospitalization. Four months later, she presented with increased bruising and fatigue. Labs showed leukocytosis, thrombocytopenia, anemia and circulating peripheral blasts. Bone marrow aspirate and biopsy was consistent with B-cell ALL. She is receiving chemotherapy on study COG AALL1732. Conclusion: Severe aplastic anemia (SAA) has high morbidity and mortality, and timely diagnosis is needed for appropriate treatment. Multiple different viral infections have been known to cause acquired aplastic anemia. Data on all the sequelae of COVID-19 infection is still emerging, but it is plausible that COVID-19 infection may cause SAA. All three patients were found to have positive COVID-19 antibodies but did not have any evidence of previous COVID-19 infection. Further research and follow-up is needed to determine if previous COVID-19 infection is indeed a risk factor for development of S A. [Formula presented] Disclosures: No relevant conflicts of interest to declare.

[Methods of SARS-CoV-2 detection in tissue]. / Nachweismethoden von SARS-CoV-2 in Gewebe.

BACKGROUND: Analyses for the presence of SARS-CoV­2 in the tissues of COVID-19 patients is important in order to improve our understanding of the disease pathophysiology for interpretation of diagnostic histopathological findings in autopsies, biopsies, or surgical specimens and to assess the potential for occupational infectious hazard. MATERIAL AND METHODS: In this review we identified 136 published studies in PubMed's curated literature database LitCovid on SARS-CoV­2 detection methods in tissues and evaluated them regarding sources of error, specificity, and sensitivity of the methods, taking into account our own experience. RESULTS: Currently, no sufficiently specific histomorphological alterations or diagnostic features for COVID-19 are known. Therefore, three approaches for SARS-CoV­2 detection are used: RNA, proteins/antigens, or morphological detection by electron microscopy. In the preanalytical phase, the dominant source of error is tissue quality, especially the different intervals between sample collection and processing or fixation (and its duration) and specifically the interval between death and sample collection in autopsies. However, this information is found in less than half of the studies (e.g., in only 42% of autopsy studies). Our own experience and first studies prove the significantly higher sensitivity and specificity of RNA-based detection methods compared to antigen or protein detection by immunohistochemistry or immunofluorescence. Detection by electron microscopy is time consuming and difficult to interpret. CONCLUSIONS: Different methods are available for the detection of SARS-CoV­2 in tissue. Currently, RNA detection by RT-PCR is the method of choice. However, extensive validation studies and method harmonization are not available and are absolutely necessary.

Detection methods for SARS-CoV-2 in tissue.

BACKGROUND: Analyses for the presence of SARS-CoV­2 in the tissues of COVID-19 patients is important in order to improve our understanding of the disease pathophysiology for interpretation of diagnostic histopathological findings in autopsies, biopsies, or surgical specimens and to assess the potential for occupational infectious hazard. MATERIAL AND METHODS: In this review we identified 136 published studies in PubMed's curated literature database LitCovid on SARS-CoV­2 detection methods in tissues and evaluated them regarding sources of error, specificity, and sensitivity of the methods, taking into account our own experience. RESULTS: Currently, no sufficiently specific histomorphological alterations or diagnostic features for COVID-19 are known. Therefore, three approaches for SARS-CoV­2 detection are used: RNA, proteins/antigens, or morphological detection by electron microscopy. In the preanalytical phase, the dominant source of error is tissue quality, especially the different intervals between sample collection and processing or fixation (and its duration) and specifically the interval between death and sample collection in autopsies. However, this information is found in less than half of the studies (e.g., in only 42% of autopsy studies). Our own experience and first studies prove the significantly higher sensitivity and specificity of RNA-based detection methods compared to antigen or protein detection by immunohistochemistry or immunofluorescence. Detection by electron microscopy is time consuming and difficult to interpret. CONCLUSIONS: Different methods are available for the detection of SARS-CoV­2 in tissue. Currently, RNA detection by RT-PCR is the method of choice. However, extensive validation studies and method harmonization are not available and are absolutely necessary.