scholarly journals Recurrent and Refractory Hemophagocytic Lymphohistiocytosis in an Elderly. Role of Immune Aberration Due to Myeloid Gene Mutation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 24-26
Author(s):  
Suthanthira kannan Ramamoorthy ◽  
Tina Noutsos ◽  
David Wei ◽  
Alexandra Yasmin Laidman ◽  
Ferenc Szabo
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Next Generation Sequencing ◽  
T Cell ◽  
Myelodysplastic Syndrome ◽  
Hemophagocytic Lymphohistiocytosis ◽  
Bone Marrow Examination ◽  
Next Generation ◽  
Tet2 Mutation ◽  
Generation Sequencing

Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal hyper-inflammatory disease induced by aberrant immune activation and subsequent proliferation of macrophages, histiocytes and T-helper cells. In this abstract we present a case of HLH, which relapsed twice despite ongoing treatment, and we hypothesize on possible causes and mechanisms. A 77 year old female presented to our hospital with ongoing fevers and worsening cytopenia. Blood counts from three years before the current presentation showed Hb 120g/L, WBC 4.0 x 10^9/L, Neutrophil count 1.8 x 10^9/L, Lymphocytes count 1.8 x 10^9/L and Platelet count 104 x 10^9/L. A bone marrow examination at that time revealed a normocellular marrow with 28% lymphocytes of which70% were CD 4+, CD 3+, CD5+ and CD 7-. Molecular studies confirmed T cell receptor (TCR) gamma gene rearrangement. The karyotype on the bone marrow was normal. In the absence of clinical symptoms, the patient was regularly followed up without specific therapy. During the current admission, however, the patient was febrile, had progressive pancytopenia and biochemistry suggestive of HLH (Fig 1). She was extensively evaluated which ruled out infective and malignant causes. A bone marrow aspirate and biopsy was performed and treatment initiated as per HLH-94 protocol. The bone marrow examination showed marked features of haemophagocytosis on a normocellular background. A small clone of T-lymphocytes was again noted with similar features as in the first biopsy. In addition, a prominent population (10%) of promonocytes was apparent with an uncertain significance. Karyotype was normal. Next Generation Sequencing showed TET2 frame shift mutation at low variant allele frequency (5%). Patient responded well to treatment. While on tapering dose of steroids, the disease flared up (Fig 1) and the patient was restarted on high dose steroids with etoposide. After a quick initial response, while still on active treatment, she again relapsed within 3 weeks, coupled with sepsis and acute myocardial ischemia, followed by sudden death. We were unable to identify a cause for HLH. There were 10% promonocytes in bone marrow and evidence of aberrant T-cells on flow cytometry. Although there was no obvious evidence of dysplasia on microscopy, the flow cytometry showed up- regulation of CD 64 and CD 14, down regulation of CD13 and 11b, and CD 34 expression in granulocytes possible indicating dysplasia as per the Wells criteria. (Wells et al., Blood 2003; 102(1):393) The Next generation sequencing showed TET2 mutation as mentioned above. Mutations in TET2 have been found to have overrepresented in chronic myelomonocytic leukemia in as much as 50% of patients and around 20-35% of patients with myelodysplastic syndrome (MDS). These patients with TET2 mutation have been found to have altered methylation. Recently TET2 has been implicated in immune regulation with evidence of abnormal CD 4 T cell proliferation (present in our patient) and disruption of T cell homeostasis. In addition, patients with TET2 mutation associated myelodysplastic syndrome are known to have auto-immune manifestations (Yimei Feng et al., Frontiers in Oncology, 2019 (9):1). Alyssa H et al have shown that TET2 mutant in patients with MDS (Myelodysplasia) leads to alteration of immune environment in the macrophage differentiation (Alyssa et al., Experimental Hematology, 2017:55; 56). Whether these immune aberration caused recurrent florid relapse of hemophagocytosis in our patient within a span of 2-3 months remained unclear and it could be considered in future research. Even though the occurrence of hemophagocytic syndrome has been described in acute leukemias, the association of the same with myeloid gene aberrations with or without overt myelodysplastic features is unknown. However since hemophagocytosis itself is not common, a careful evaluation to look for uncommon associations which may be a triggering factor may pave the way for identifying their possible role in the pathogenesis. And therapeutic options such as hypo methylating agents can evolve when such associations are confirmed in future studies. Figure 1 Disclosures No relevant conflicts of interest to declare.

Mutation Analysis in 200 Cases of Newly Diagnosed Myelodysplastic Syndrome By Next Generation Sequencing: Association with Established Prognostic Variables and IPSS-R

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1703-1703
Author(s):  
Kankana Ghosh ◽  
Parsa Hodjat ◽  
Priyanka Priyanka ◽  
Beenu Thakral ◽  
Keyur P. Patel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Next Generation Sequencing ◽  
Myelodysplastic Syndrome ◽  
Mutation Analysis ◽  
Conflicts Of Interest ◽  
International Prognostic Scoring System ◽  
P Value ◽  
Newly Diagnosed ◽  
Next Generation ◽  
Generation Sequencing

Abstract INTRODUCTION Myelodysplastic syndrome (MDS) is known to have numerous genomic aberrations that predict response to treatment and overall survival. We aimed to assess various mutations in newly diagnosed MDS cases by next generation sequencing (NGS) and their association with various well-established clinicopathologic parameters and the Revised International Prognostic Scoring System (IPSS-R). MATERIALS AND METHODS We performed molecular studies on DNA extracted from bone marrow aspirate specimens in 200 newly diagnosed treatment naïve MDS patients presenting at a single institution from 08/2013 to 03/2015 as part of routine clinical work up in a CLIA certified molecular diagnostics laboratory. Cases met criteria for MDS per WHO 2008 criteria. The entire coding sequences of 28 genes (ABL1, ASXL1, BRAF, DNMT3A, EGFR, EZH2, FLT3, GATA1, GATA2, HRAS, IDH1, IDH2, IKZF2, JAK2, KIT, KRAS, MDM2, MLL, MPL, MYD88, NOTCH1, NPM1, NRAS, PTPN11, RUNX1, TET2, TP53, WT1) were sequenced using a NGS-based custom-designed assay using TruSeq chemistry on Illumina MiSeq platform. FLT3 internal tandem duplications (ITD) and codon 835/836 point mutation were detected by PCR followed by capillary electrophoresis. CEBPA mutation analysis was performed by PCR followed by Sanger sequencing on 186 patients. RESULTS Median age was 67 years. Patients included 139 males (69.5%) and 61 females (30.5%). Hematologic parameters are as follows [median (range)]: Hb 9.6 g/dL (5-16.7), platelets 75 K/μ L (5-652), WBC: 2.8 K/μ L (0.4-20.8), ANC 1.3 K/μ L (0.0 -12.0), AMC 0.2 K/μ L (0.0-3). Bone marrow (BM) blasts [median (range)] were 4% (0-19). Of 192 patients with cytogenetic analysis performed, 65 (33.85%) had diploid karyotype, 53 (27.6%) had one, 21 (10.93%) had two, 13 (6.77%) had three, 40 (20.83%) had > three abnormalities. IPSS-R risk categorization of the 200 cases is as follows: very low (17 cases, 8.5%), low (46, 23%) intermediate (42, 21%), high (47, 23.5%), very high (48, 24%). Mutations identified by NGS are as detailed in Table 1. Of the 4 patients with FLT mutations detected, the breakdown is as follows: FLT3 ITD (3, 75%), FLT3 D835 (1, 25%), FLT3, ITD + D835 (0, 0%). CEBPA mutation was detected in 12 of 186 (6.45%) cases assessed. CEBPA was detected in 12 (6.45%). Sixty three (31.5%) cases had no mutations detected in the genes analyzed by NGS or PCR, 80 (40%) had mutations in one, 42 (21%) had mutations in two, 8 (4%) in three and 7 (3.5%) in > three genes. We found positive associations between mutated genes and various parameters as detailed in Table 2. No association was found between frequency of any particular mutation and the IPSS-R score. CONCLUSIONS: MDS is a heterogeneous group of myeloid neoplasms at the genetic level. Multiple genetic mutations in a large subset of cases likely indicate clonal evolution. A subset of mutations has significant association with well-established clinico-pathologic parameters like WBC and BM blast percentage. With longer follow-up, we could use this data to refine IPSS-R. Table 1. Number of cases % cases TP53 46 23 TET2 33 16.5 RUNX1 27 13.5 ASXL1 25 12.5 DNMT3A 17 8.5 EZH2 12 6 IDH2 8 4 IDH1 7 3.5 NRAS 7 3.5 JAK2 5 2.5 FLT3 4 2 PTPN11 3 1.5 EGFR 2 1 MPL 2 1 WT1 2 1 GATA2 1 0.5 KIT 1 0.5 KRAS 1 0.5 MYD88 1 0.5 NPM1 1 0.5 BRAF 1 0.5 Table 2. Mutated genes p value WBC ASXL1 <0.042 AEC TET2 <0.016 BM blast % RUNX1, CEBPA <0.008, p<0.02 BM myelocyte % TP53, TET2, RUNX1, DNMT3A <0.014, <0.014, <0.015, <0.038 AEC: absolute eosinophil count, BM: bone marrow Disclosures No relevant conflicts of interest to declare.

scholarly journals Role of Next Generation Sequencing in the Diagnosis of Unexplained Thrombocytopenias- an Experience at a Tertiary Care Hospital

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1017-1017
Author(s):  
Karthika K V ◽  
Mobin Paul ◽  
Anjana K V ◽  
Ullas Mony ◽  
Ganeshprasad Arunkumar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Next Generation Sequencing ◽  
Family History ◽  
Bone Marrow Failure ◽  
Positive Family History ◽  
Bone Marrow Examination ◽  
Care Hospital ◽  
Next Generation ◽  
Inherited Disorders ◽  
Generation Sequencing

Abstract BACKGROUND Next Generation Sequencing (NGS) has been enormously rewarding in the field of diagnostic hematology. In particular, the diagnosis of inherited disorders has progressed in leaps and bounds. These patients tend to remain undiagnosed for a long period of time not only because of unavailability of molecular diagnostics but also due to lack of cognizance and atypical presentations. Thrombocytopenia (TCP) is a common hematological presentation and can lead to chronic hospital visits to life-threatening bleeds. Most of these patients have acquired disorders such as immune TCP, malignancies, liver disease etc. However, some of them are likely to have unidentified inherited causes. We thus intended to study the utility of NGS in the definitive diagnosis of unexplained TCPs with or without other cytopenias to understand the clinicopathologic characteristics of these patients. METHODOLOGY This was a retrospective descriptive study done at two centres over three years from May 2018 to May 2021. Patients with TCP with one of the following: (a) positive family history (b) clinical/ laboratory clues to an inherited cause (c) chronic TCP with no response to conventional therapies and sent for clinical exome sequencing done by NGS were included in the study. Patients who were negative for germline mutations were excluded. Sequencing of targeted genes was performed on the Illumina platform with a mean coverage of &gt;80-100X. Mutations annotated as pathogenic, likely pathogenic and variant of uncertain significance (VUS) were considered clinically significant. VUS are mutations that are difficult to classify as pathogenic and require clinical validation and family testing. RESULTS Our cohort included 18 patients and were divided into two groups- cases of isolated TCP and cases of TCP with anemia and/or neutropenia. Patients presenting with isolated thrombocytopenia We had nine cases of isolated TCP out of which there were three cases of X-linked macrothrombocytopenia with MYH9 mutation, two cases of Wiskott Aldrich syndrome (WAS) and one case each of congenital thrombotic thrombocytopenic purpura (TTP), atypical Hemolytic Uremic Syndrome (HUS), Fanconi anemia (FA) and grey platelet syndrome. The demographic and mutational characteristics are described in Table 1. Clinical and laboratory clues were present in 7 cases, such as chronic kidney disease, micro/ macrothrombocytopenia, neutrophil inclusions etc. Bone marrow examination was carried out in 4 cases- the significant dyspoiesis in FA and myelofibrosis in grey platelet syndrome mislead to a diagnosis of MDS and myelofibrosis respectively. Seven patients had received treatment with steroids, immunosuppressants, splenectomy, danazol and TPO mimetics before the NGS diagnosis. Patients presenting with thrombocytopenia and other cytopenias This group consisted of nine cases with two cases each of Dyskeratosis Congenita (DKC) and WAS and one case each of TTP, Ghosal hematodiaphyseal dysplasia, Congenital Amegakaryocytic Thrombocytopenia (CAMT), B-cell immunodeficiency with hypogammaglobulinemia type-25 and double homozygous for FA and DKC. The majority of patients in this group were young and had lower platelet counts (Table 2). The most common associated cytopenia was anemia. Phenotypic clues to diagnosis were present in cases of DKC and WAS. The common differentials considered in this group were inherited bone marrow failure syndromes (IBMFS), congenital immunodeficiency syndromes etc. Bone marrow examination was done more frequently in these patients and showed hypocellular marrow in IBMFS and absent megakaryocytes in CAMT. These patients have also been treated with steroids, IVIg, danazol and TPO mimetics. CONCLUSION Patients with inherited isolated TCPs have a chronic course and heterogenous causes therefore tend to be diagnosed later in life. However, patients with TCPs and other cytopenias tend to present at a younger age with infrequent family history. IBMFS was the most common disorder identified in this latter group of patients. Positive family history, clinical and laboratory clues and absence of response to conventional therapies should prompt workup of inherited causes by NGS to avoid long term ineffectual treatment. Further, NGS mutations, in particular VUS have to be interpreted with caution with the help of parental study, clinical presentation, in-silico analysis and inputs from molecular and genetic experts. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Immortalization and Functional Screening of Natively Paired Human T Cell Receptor Repertoires

2021 ◽  
Author(s):  
Ahmed S Fahad ◽  
Cheng Yu Chung ◽  
Sheila N. Lopez Acevedo ◽  
Nicoleen Boyle ◽  
Bharat Madan ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Functional Screening ◽  
Next Generation ◽  
Sequencing Technologies ◽  
Human T Cell ◽  
Screening Platform ◽  
Generation Sequencing

Functional analyses of the T cell receptor (TCR) landscape can reveal critical information about protection from disease and molecular responses to vaccines. However, it has proven difficult to combine advanced next-generation sequencing technologies with methods to decode the peptide-major histocompatibility complex (pMHC) specificity of individual TCRs. Here we developed a new high-throughput approach to enable repertoire-scale functional evaluations of natively paired TCRs. In particular, we leveraged the immortalized nature of physically linked TCRα:β amplicon libraries to analyze binding against multiple recombinant pMHCs on a repertoire scale. To exemplify the utility of this approach, we also performed affinity-based functional mapping in conjunction with quantitative next-generation sequencing to track antigen- specific TCRs. These data successfully validated a new immortalization and screening platform to facilitate detailed molecular analyses of human TCRs against diverse antigen targets associated with health, vaccination, or disease.

scholarly journals A rare atypical chronic myeloid leukemia BCR-ABL1 negative with concomitant JAK2 V617F and SETBP1 mutations: a case report and literature review

2020 ◽  
Vol 11 ◽  
pp. 204062072092710
Author(s):  
Tianqi Gao ◽  
Changhui Yu ◽  
Si Xia ◽  
Ting Liang ◽  
Xuekui Gu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Targeted Therapy ◽  
Next Generation Sequencing ◽  
Myeloid Leukemia ◽  
Jak2 V617f ◽  
Next Generation ◽  
Bone Marrow Fibrosis ◽  
Atypical Chronic Myeloid Leukemia ◽  
Generation Sequencing ◽  
Marrow Fibrosis

Atypical chronic myeloid leukemia (aCML) BCR-ABL1 negative is a rare myelodysplastic syndromes/myeloproliferative neoplasm (MDS/MPN) for which no standard treatment currently exists. The advent of next-generation sequencing has allowed our understanding of the molecular pathogenesis of aCML to be expanded and has made it possible for clinicians to more accurately differentiate aCML from similar MDS/MPN overlap syndrome and MPN counterparts, as MPN-associated driver mutations in JAK2, CALR, or MPL are typically absent in aCML. A 55-year old male with main complaints of weight loss and fatigue for more than half a year and night sweats for more than 2 months was admitted to our hospital. Further examination revealed increased white blood cells, splenomegaly, and grade 1 bone marrow fibrosis with JAK2 V617F, which supported a preliminary diagnosis of pre-primary marrow fibrosis. However, in addition to JAK2 V617F (51.00%), next-generation sequencing also detected SETBP1 D868N (46.00%), ASXL1 G645fs (36.09%), and SRSF2 P95_R102del (33.56%) mutations. According to the 2016 World Health Organization diagnostic criteria, the patient was ultimately diagnosed with rare aCML with concomitant JAK2 V617F and SETBP1 mutations. The patient received targeted therapy of ruxolitinib for 5 months and subsequently an additional four courses of combined hypomethylating therapy. The patient exhibited an optimal response, with decreased spleen volume by approximately 35% after therapy and improved symptom scores after therapy. In diagnosing primary bone marrow fibrosis, attention should be paid to the identification of MDS/MPN. In addition to basic cell morphology, mutational analysis using next-generation sequencing plays an increasingly important role in the differential diagnosis. aCML with concomitant JAK2 V617F and SETBP1 mutations has been rarely reported, and targeted therapy for mutated JAK2 may benefit patients, especially those not suitable recipients of hematopoietic stem cell transplants.

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